Adenoviral gene therapy vectors and methods of use thereof

Abstract

Among other things, the present disclosure provides adenoviral vectors, genomes, and methods of use thereof. In some embodiments, the present disclosure provides methods of in vivo gene therapy comprising administration of an adenoviral payload vector and an adenoviral integration vector. The present disclosure provides, among other things, compositions comprising an adenoviral payload vector and an adenoviral integration vector for use in, e.g., methods of in vivo gene therapy. In some embodiments, the present disclosure provides adenoviral vectors, genomes, and compositions thereof comprising a therapeutic sequence, e.g., a nucleic acid sequence encoding a CYBB therapeutic polypeptide. In some embodiments, the present disclosure provides methods of treating various conditions, disorders, and diseases, e.g., chronic granulomatous disease.

Description

ADENOVIRAL GENE THERAPY VECTORS AND METHODS OF USE THEREOF CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Nos. 63 / 701,547, filed September 30, 2024, 63 / 718,975, filed November 11, 2024, and 63 / 787,654, filed April 11, 2025, the entirety of each of which is incorporated herein by reference. BACKGROUND

[0002] Many medical conditions have the potential to be treated or ameliorated by gene therapy. Adenoviral vectors can be used in gene therapy, including in vivo gene therapy. Compositions and methods that facilitate adenoviral vector-based gene therapy are therefore needed. SUMMARY

[0003] Among other things, the present disclosure provides methods of in vivo gene therapy. In some embodiments, a method of in vivo gene therapy comprises administering to the subject a dose of a pharmaceutical composition comprising a helper dependent adenoviral (HDAd) payload vector, e.g., a HDAd payload vector provided herein, and a helper dependent adenoviral (HDAd) integration vector, e.g., a HDAd integration vector provided herein. In some embodiments, a method of in vivo gene therapy comprises administering to the subject a dose of a pharmaceutical composition comprising a HDAd payload vector, e.g., a HDAd payload vector provided herein, and a HDAd integration vector, e.g., a HDAd integration vector provided herein, wherein the HDAd payload vector and HDAd integration vector are at a ratio of about 1-3:1 or 1:1-3 HDAd payload vector:HDAd integration vector. In some embodiments, a method of in vivo gene therapy comprises administering to the subject a dose of a pharmaceutical composition comprising a HDAd payload vector, e.g., a HDAd payload vector provided herein, and a HDAd integration vector, e.g., a HDAd integration vector provided herein, wherein the HDAd payload vector and HDAd integration vector are at a ratio of about 1-3:1 or 1:1-3 HDAd payload vector:HDAd integration vector and wherein a dose is about 2.5 x 1011GC / kg to about 1.25 x 1013GC / kg. In some embodiments, a method of in vivo gene therapy comprises administering to the subject a dose of a pharmaceutical composition comprising a HDAd payload vector and a HDAd integration vector, wherein the HDAd payload vector and HDAd integration vector are at a ratio of about 1-3:1 or 1:1-3 HDAd payload vector:HDAd integration vector; wherein a dose is about 2.5 x 1011GC / kg to about 1.25 x 1013GC / kg; wherein the HDAd payload vector comprises a CD46-binding adenoviral capsid and a double-stranded DNA genome comprising (1) a 5’ ITR and a 3’ ITR; (2) a packaging sequence; (3) a first transposase target site and a second transposase target site; (4) a payload cassette comprising (a) a therapeutic sequencecomprising a nucleic acid sequence encoding a therapeutic polypeptide, wherein the therapeutic polypeptides is a CYBB, CYBA, NCF1, NCF2, NCF4, or CYBC1 therapeutic polypeptide; and (b) a promoter operably linked to the therapeutic sequence; and (5) a selection cassette comprising (a) a nucleic acid sequence encoding a selection marker; and (b) a promoter operably linked to the nucleic acid sequence encoding the selection marker; wherein (i) the payload cassette and the selection cassette are located between the first transposase target site and the second transposase target site; and (ii) the packaging sequence is located 5’ to the first transposase target site; and wherein the HDAd integration vector comprises a CD46-binding adenoviral capsid and a double-stranded DNA genome comprising (1) a 5’ ITR and a 3’ ITR; (2) a packaging sequence; (3) a nucleic acid sequence encoding a transposase.

[0004] In some embodiments, a subject is suffering from a condition, disorder, or disease. In some embodiments, a method of in vivo gene therapy comprises treating a condition, disorder, or disease. In some embodiments, a subject is suffering from a condition, disorder, or disease and a method of in vivo gene therapy comprises treating a condition, disorder, or disease. In some embodiments, a condition, disorder, or disease is an immune deficiency disorder. In some embodiments, a subject is suffering from a defect in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. In some embodiments, a condition, disorder, or disease is chronic granulomatous disease (CGD). In some embodiments, a condition, disorder, or disease is X-linked CGD. In some embodiments, a condition, disorder, or disease is autosomal recessive CGD.

[0005] In some embodiments, a CD46-binding adenoviral capsid of a HDAd payload vector is an Ad5 / 35 capsid. In some embodiments, a CD46-binding adenoviral capsid of a HDAd payload vector is an Ad5 / 35++ capsid. In some embodiments, a CD46-binding adenoviral capsid of a HDAd integration vector is an Ad5 / 35 capsid. In some embodiments, a CD46-binding adenoviral capsid of a HDAd integration vector is an Ad5 / 35++ capsid.

[0006] In some embodiments, a therapeutic sequence comprises or is a nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments, a therapeutic sequence comprises or is a nucleic acid sequence encoding a therapeutic nucleic acid. In some embodiments, a nucleic acid sequence encoding a therapeutic polypeptide is codon-optimized. In some embodiments, a therapeutic polypeptide comprises or is a CYBB therapeutic polypeptide. In some embodiments, a therapeutic polypeptide comprises or is a CYBA therapeutic polypeptide. In some embodiments, a therapeutic polypeptide comprises or is a NCF1 therapeutic polypeptide. In some embodiments, a therapeutic polypeptide comprises or is a NCF2 therapeutic polypeptide. In some embodiments, a therapeutic polypeptide comprises or is a NCF4 therapeutic polypeptide. In some embodiments, a therapeutic polypeptide comprises or is a CYBC1 therapeutic polypeptide. In some embodiments, a nucleic acid sequence encoding a therapeutic polypeptide comprises a nucleic acid sequence that has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more identity to the nucleic acid sequence of SEQ ID NO: 79. In some embodiments, a nucleic acid sequence encoding a therapeutic polypeptide comprises or is the nucleic acid sequence of SEQ ID NO: 79. In some embodiments, a therapeutic polypeptide comprises or is the amino acid sequence of any one of SEQ ID NOs: 151-161.

[0007] In some embodiments, a 5’ ITR of a genome of a HDAd payload vector is an Ad55’ ITR. In some embodiments, a 3’ ITR of a genome of a HDAd payload vector is an Ad5 3’ ITR. In some embodiments, a 5’ ITR of a genome of a HDAd payload vector is an Ad55’ ITR and a 3’ ITR of a genome of a HDAd payload vector is an Ad53’ ITR. In some embodiments, a packaging sequence of a genome of a HDAd payload vector is an Ad5 packaging sequence.

[0008] In some embodiments, a promoter operably linked to a therapeutic sequence comprises or is a lineage-specific promoter. In some embodiments, a promoter operably linked to a therapeutic sequence comprises or is a ubiquitous promoter. In some embodiments, a promoter operably linked to a therapeutic sequence comprises or is a CTSG-FES promoter. In some embodiments, a promoter operably linked to a therapeutic sequence comprises or is the nucleic acid sequence of any one of SEQ ID NOs: 88-106 and 175.

[0009] In some embodiments, a payload cassette comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of SEQ ID NO: 80. In some embodiments, a payload cassette comprises or is the nucleic acid sequence of SEQ ID NO: 80.

[0010] In some embodiments, a genome of a HDAd payload vector comprises a chromatin insulator. In some embodiments, a chromatin insulator comprises or is the nucleic acid sequence of SEQ ID NO: 133.

[0011] In some embodiments, a selection marker is MGMTP140K. In some embodiments, a nucleic acid sequence encoding a selection marker comprises or is the nucleic acid sequence of SEQ ID NO: 81. In some embodiments, a selection marker is a CD117 polypeptide comprising one or more mutations.

[0012] In some embodiments, a promoter operably linked to a nucleic acid sequence encoding a selection marker comprises or is a ubiquitous promoter. In some embodiments, a promoter operably linked to a nucleic acid sequence encoding a selection marker comprises or is an EF1a, ACTB, EFS, eIF4A1, CAG, CMV, GAPDH, KIN, PGK, ROSA, SFFV, SV40, UBB, or UBC promoter. In some embodiments, a promoter operably linked to a nucleic acid sequence encoding a selection marker comprises or is an EF1a promoter. In some embodiments, a promoter operably linked to a nucleic acid sequence encoding a selection marker comprises or is the nucleic acid sequence of any one of SEQ ID NOs: 107-127, 171, and 172.

[0013] In some embodiments, a selection cassette comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of SEQ ID NO: 83. In some embodiments, a selection cassette comprises or is the nucleicacid sequence of SEQ ID NO: 83.

[0014] In some embodiments, a first transposase target site is a Sleeping Beauty (SB) transposase target site and a second transposase target site is a SB transposase target site.

[0015] In some embodiments, a genome of a HDAd payload vector comprises a first recombinase target site and a second recombinase target site. In some embodiments, a genome of a HDAd payload vector comprises a first recombinase target site and a second recombinase target site, wherein a first transposase target site and a second transposase target site are located between the first recombinase target site and the second recombinase target site. In some embodiments, a genome of a HDAd payload vector comprises a first recombinase target site and a second recombinase target site, wherein a packaging sequence is located 5’ to the first recombinase target site. In some embodiments, a genome of a HDAd payload vector comprises a first recombinase target site and a second recombinase target site, wherein a first transposase target site and a second transposase target site are located between the first recombinase target site and the second recombinase target site and a packaging sequence is located 5’ to the first recombinase target site. In some embodiments, a first recombinase target site is a Flp recombinase target site and a second recombinase target site is a Flp recombinase target site. In some embodiments, a first recombinase target site is a Flpx9 or FlpE recombinase target site and a second recombinase target site is a Flpx9 or FlpE recombinase target site.

[0016] In some embodiments, a genome of a HDAd payload vector comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of SEQ ID NO: 134. In some embodiments, a genome of the HDAd payload vector comprises or is the nucleic acid sequence of SEQ ID NO: 134.

[0017] In some embodiments, a transposase comprises or is a Sleeping Beauty (SB) transposase. In some embodiments, a SB transposase is a SB10 transposase, SB100X transposase, SB100X+ transposase, or M3A transposase or a variant thereof. In some embodiments, a transposase comprises or is the amino acid sequence of SEQ ID NO: 135-138.

[0018] In some embodiments, a genome of an HDAd integration vector comprises a nucleic acid sequence encoding a recombinase. In some embodiments, a recombinase comprises or is a Flp recombinase. In some embodiments, a Flp recombinase comprises one or more mutations, optionally wherein one or more mutations are selected from P2S, L33S, Y108N, S294P, or a combination thereof. In some embodiments, a Flp recombinase comprises or is a Flpx9 recombinase, FlpE recombinase, or FlpO recombinase. In some embodiments, a recombinase comprises or is the amino acid sequence of any one of SEQ ID NOs: 139-141.

[0019] In some embodiments, a 5’ ITR of a genome of a HDAd integration vector is an Ad55’ ITR. In some embodiments, a 3’ ITR of a genome of a HDAd integration vector is an Ad53’ ITR. In someembodiments, a 5’ ITR of a genome of a HDAd integration vector is an Ad55’ ITR and a 3’ ITR of a genome of a HDAd integration vector is an Ad53’ ITR. In some embodiments, a packaging sequence of a genome of a HDAd integration vector is an Ad5 packaging sequence.

[0020] In some embodiments, a genome of the HDAd integration vector comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of any one of SEQ ID NOs: 144-146. In some embodiments, a genome of the HDAd integration vector comprises or is the nucleic acid sequence of any one of SEQ ID NOs: 144-146.

[0021] In some embodiments, a pharmaceutical composition is a liquid composition. In some embodiments, a pharmaceutical composition comprises a buffer. In some embodiments, a pharmaceutical composition is administered intravenously. In some embodiments, a HDAd payload vector and a HDAd integration vector are at ratio of about 1-3:1 or 1:1-3 HDAd payload vector:HDAd integration vector. In some embodiments, no more than one dose of a pharmaceutical composition is administered to a subject. In some embodiments, a dose is about 2.5 x 1011GC / kg to about 1.25 x 1013GC / kg.

[0022] In some embodiments, a method of in vivo gene therapy comprises mobilizing hematopoietic stem cells of a subject prior to administering a HDAd payload vector. In some embodiments, mobilizing hematopoietic stem cells of a subject comprises administering to a subject granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), plerixafor, motixafortide, stem cell factor (SCF), tGRO-beta, and / or a VLA4-targeting agent. In some embodiments, G-CSF and plerixafor are administered to a subject. In some embodiments, tGRO-beta and plerixafor are administered to a subject. In some embodiments, G-CSF and motixafortide are administered to a subject.

[0023] In some embodiments, G-CSF is administered to a subject daily. In some embodiments, G- CSF is administered to a subject for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days. In some embodiments, G-CSF is administered to a subject for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days prior to administration of a HDAd payload vector. In some embodiments, G-CSF is administered to a subject on a day of administration of a HDAd payload vector.

[0024] In some embodiments, plerixafor is administered to a subject daily. In some embodiments, plerixafor is administered to a subject for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days. In some embodiments, plerixafor is administered to a subject for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days prior to administration of a HDAd payload vector. In some embodiments, plerixafor is administered to a subject on a day of administration of a HDAd payload vector.

[0025] In some embodiments, a method of in vivo gene therapy comprises administering to a subject an immunosuppression regimen comprising one or more immunosuppression agents.

[0026] In some embodiments, an immunosuppression regimen comprises a corticosteroid. In someembodiments, a corticosteroid comprises or is a glucocorticoid. In some embodiments, a glucocorticoid comprises or is dexamethasone, prednisone, prednisolone, or methylprednisolone. In some embodiments, a corticosteroid is administered to a subject daily. In some embodiments, a corticosteroid is administered to a subject for at least about 1, 2, 3, 4, 5, 6, 7 or more days or at least about 1, 2 or more weeks. In some embodiments, a corticosteroid is administered to a subject for at least about 1, 2, 3, 4, 5 or more days prior to administration of a HDAd payload vector. In some embodiments, a corticosteroid is administered to a subject on a day of administration of a HDAd payload vector.

[0027] In some embodiments, an immunosuppression regimen comprises an inflammatory signal inhibitor. In some embodiments, an inflammatory signal inhibitor comprises or is an interleukin-1 signal inhibitor. In some embodiments, an interleukin-1 signal inhibitor comprises or is an interleukin-1 receptor antagonist. In some embodiments, an interleukin-1 receptor antagonist comprises or is anakinra. In some embodiments, an inflammatory signal inhibitor is administered to a subject daily. In some embodiments, an inflammatory signal inhibitor is administered to a subject for at least about 1, 2, 3, 4, 5 or more days. In some embodiments, an inflammatory signal inhibitor is administered to a subject for at least about 1, 2, 3, 4, 5 or more days prior to administration of a HDAd payload vector.

[0028] In some embodiments, an immunosuppression regimen comprises an interleukin-6 receptor antagonist. In some embodiments, an inteleukin-6 receptor antagonist comprises or is tocilizumab or siltuximab. In some embodiments, an interleukin-6 receptor antagonist is administered to a subject daily. In some embodiments, an interleukin-6 receptor antagonist is administered to a subject for at least about 1, 2, 3, 4, 5 or more days. In some embodiments, an interleukin-6 receptor antagonist is administered to a subject for at least about 1, 2, 3, 4, 5 or more days prior to administration of a HDAd payload vector. In some embodiments, an interleukin-6 receptor antagonist is administered to a subject on a day of administration of a HDAd payload vector.

[0029] In some embodiments, an immunosuppression regimen comprises an anti-IL-17 antibody, an anti-IL-23 antibody, an anti-TNFa antibody, and / or an anti-IFNg antibody, optionally wherein the anti- IFNg antibody comprises or is emapalumab.

[0030] In some embodiments, a method of in vivo gene therapy comprises administering to a subject one or more selection agents. In some embodiments, a selection marker is MGMTP140Kand one or more selection agents comprise temozolomide (TMZ), O6-benzylguanine (O6BG), and / or 1,3-bis(2- chloroethyl)-1-nitrosourea (BCNU). In some embodiments, TMZ and O6BG, BCNU and O6BG, or TMZ and BCNU are administered to a subject.

[0031] In some embodiments, TMZ is administered to a subject orally or intravenously. In some embodiments, TMZ is administered to a subject about once every 28 days or about monthly. In some embodiments, 1, 2, 3, 4, 5 or more doses of TMZ are administered to a subject. In some embodiments, afirst dose of TMZ is administered to a subject at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks following administration of a HDAd payload vector. In some embodiments, TMZ is administered to a subject at a dose of about 300-600 mg / m2.

[0032] In some embodiments, O6BG is administered to a subject intravenously. In some embodiments, O6BG is administered to a subject as an intravenous bolus and / or intravenous infusion. In some embodiments, an intravenous bolus of O6BG is administered prior to an intravenous infusion of O6BG. In some embodiments, O6BG is administered to a subject about once every 28 days or about monthly. In some embodiments, 1, 2, 3, 4, 5 or more doses of O6BG are administered to a subject. In some embodiments, a first dose of O6BG is administered to a subject at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks following administration of a HDAd payload vector. In some embodiments, O6BG is administered to the subject at a dose of about 80-280 mg / m2. In some embodiments, a dose of O6BG comprises an intravenous bolus dose of about 70-170 mg / m2O6BG and / or an intravenous infusion dose of about 10-110 mg / m2.

[0033] In some embodiments, BCNU is administered to a subject orally or intravenously. In some embodiments, BCNU is administered to a subject about once every 28 days or about monthly. In some embodiments, 1, 2, 3, 4, 5 or more doses of BCNU are administered to a subject. In some embodiments, a first dose of BCNU is administered to a subject at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks following administration of a HDAd payload vector. In some embodiments, BCNU is administered to a subject at a dose of about 100-500 mg / m2.

[0034] In some embodiments, a selection marker is CD117 comprising one or more mutations and one or more selection agents comprise an anti-CD117 antibody or antigen binding fragment thereof. In some embodiments, an anti-CD117 antibody or antigen binding fragment thereof selectively binds to CD117 not comprising one or more mutations.

[0035] In some embodiments, a subject is a mammal, e.g., a mouse, a rat, a non-human primate. In some embodiments, a subject is a human. In some embodiments, a subject is a patient. In some embodiments, a subject is an adult. In some embodiments, a subject is a child. In some embodiments, a subject is a male. In some embodiments, a subject is a female.

[0036] In some embodiments, a subject comprises one or more mutations of a CYBB, CYBA, NCF1, NCF2, NCF4, and / or CYBC1 gene. In some embodiments, one or more mutations of a CYBB, CYBA, NCF1, NCF2, NCF4, and / or CYBC1 gene comprise a point mutation. In some embodiments, one or more mutations of a CYBB, CYBA, NCF1, NCF2, NCF4, and / or CYBC1 gene comprise a missense mutation. In some embodiments, one or more mutations of a CYBB, CYBA, NCF1, NCF2, NCF4, and / or CYBC1 gene comprise a nonsense mutation. In some embodiments, one or more mutations of a CYBB, CYBA, NCF1, NCF2, NCF4, and / or CYBC1 gene comprise a deletion. In some embodiments, one or moremutations of a CYBB, CYBA, NCF1, NCF2, NCF4, and / or CYBC1 gene comprise a frameshift mutation.

[0037] In some embodiments, a method of in vivo gene therapy reduces a symptom of a condition, disorder, or disease. In some embodiments, a symptom of a condition, disorder, or disease is reduced. In some embodiments, a symptom of a condition, disorder, or disease comprises or is a bacterial or fungal infection, optionally wherein the infection is recurrent. In some embodiments, a symptom of a condition, disorder, or disease comprises or is an abscess. In some embodiments, a symptom of a condition, disorder, or disease comprises or is a granuloma. In some embodiments, a symptom of a condition, disorder, or disease comprises or is abdominal pain, optionally wherein the abdominal pain is chronic. In some embodiments, a symptom of a condition, disorder, or disease comprises or is liver dysfunction. In some embodiments, a symptom of a condition, disorder, or disease comprises or is lung dysfunction. In some embodiments, a symptom of a condition, disorder, or disease comprises or is an autoimmune condition, optionally wherein the autoimmune condition comprises or is inflammatory bowel disease, autoimmune pulmonary disease, or lupus. In some embodiments, a symptom of a condition, disorder, or disease comprises or is lymph node swelling.

[0038] In some embodiments, a method of in vivo gene therapy independently improves one or more clinical assessments of a subject. In some embodiments, one or more clinical assessments of a subject are independently improved. In some embodiments, a clinical assessment is a level of NADPH oxidase activity in a sample from the subject and an improvement is an increase as compared to baseline. In some embodiments, level of NADPH oxidase activity is assessed in a sample from a subject by a nitroblue tetrazolium assay or dihydrorhodamine flow cytometry assay. In some embodiments, baseline is a level of NADPH oxidase activity in a sample from a subject prior to administration of a HDAd payload vector. In some embodiments, a clinical assessment is a level of DHR+ neutrophils in a sample from a subject and an improvement is an increase as compared to baseline. In some embodiments, level of DHR+ neutrophils is assessed in a sample from a subject by a dihydrorhodamine flow cytometry assay. In some embodiments, level of DHR+ neutrophils is increased by at least about 1-100%, 1-75%, 1-50%, 1-25%, 5-100%, 5-75%, 5-50%, 5-25%, 5-20%, 10-100%, 10-75%, 10-50%, 10-25%, 10-20% or more as compared to baseline. In some embodiments, level of DHR+ neutrophils is increased by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more as compared to baseline. In some embodiments, baseline is a level of DHR+ neutrophils in a sample from a subject prior to administration of a HDAd payload vector. In some embodiments, a clinical assessment is a level of expression of a therapeutic polypeptide in a sample from a subject and an improvement is an increase as compared to baseline. In some embodiments, a level of expression of a therapeutic polypeptide is assessed in a sample from a subject by flow cytometry. In some embodiments, baseline is a level of expression of atherapeutic polypeptide in a sample from a subject prior to administration of a HDAd payload vector.

[0039] In another aspect, the present disclosure provides a HDAd payload vector, e.g., a HDAd payload vector provided herein. In some embodiments, a HDAd payload vector comprises a CD46-binding adenoviral capsid and a double-stranded DNA genome comprising (1) a 5’ ITR and a 3’ ITR; (2) a packaging sequence; (3) a first transposase target site and a second transposase target site; (4) a payload cassette comprising (a) a therapeutic sequence comprising a nucleic acid sequence encoding a therapeutic polypeptide, wherein the therapeutic polypeptides is a CYBB, CYBA, NCF1, NCF2, NCF4, or CYBC1 therapeutic polypeptide; and (b) a promoter operably linked to the therapeutic sequence; and (5) a selection cassette comprising (a) a nucleic acid sequence encoding a selection marker; and (b) a promoter operably linked to the nucleic acid sequence encoding the selection marker; wherein (i) the payload cassette and the selection cassette are located between the first transposase target site and the second transposase target site; and (ii) the packaging sequence is located 5’ to the first transposase target site.

[0040] In some embodiments, a CD46-binding adenoviral capsid of a HDAd payload vector is an Ad5 / 35 capsid. In some embodiments, a CD46-binding adenoviral capsid of a HDAd payload vector is an Ad5 / 35++ capsid.

[0041] In some embodiments, a therapeutic sequence comprises or is a nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments, a therapeutic sequence comprises or is a nucleic acid sequence encoding a therapeutic nucleic acid. In some embodiments, a nucleic acid sequence encoding a therapeutic polypeptide is codon-optimized. In some embodiments, a therapeutic polypeptide comprises or is a CYBB therapeutic polypeptide. In some embodiments, a therapeutic polypeptide comprises or is a CYBA therapeutic polypeptide. In some embodiments, a therapeutic polypeptide comprises or is a NCF1 therapeutic polypeptide. In some embodiments, a therapeutic polypeptide comprises or is a NCF2 therapeutic polypeptide. In some embodiments, a therapeutic polypeptide comprises or is a NCF4 therapeutic polypeptide. In some embodiments, a therapeutic polypeptide comprises or is a CYBC1 therapeutic polypeptide. In some embodiments, a nucleic acid sequence encoding a therapeutic polypeptide comprises a nucleic acid sequence that has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the nucleic acid sequence of SEQ ID NO: 79. In some embodiments, a nucleic acid sequence encoding a therapeutic polypeptide comprises or is the nucleic acid sequence of SEQ ID NO: 79. In some embodiments, a therapeutic polypeptide comprises or is the amino acid sequence of any one of SEQ ID NOs: 151-161.

[0042] In some embodiments, a 5’ ITR of a genome of a HDAd payload vector is an Ad55’ ITR. In some embodiments, a 3’ ITR of a genome of a HDAd payload vector is an Ad5 3’ ITR. In some embodiments, a 5’ ITR of a genome of a HDAd payload vector is an Ad55’ ITR and a 3’ ITR of a genome of a HDAd payload vector is an Ad53’ ITR. In some embodiments, a packaging sequence of a genome ofa HDAd payload vector is an Ad5 packaging sequence.

[0043] In some embodiments, a promoter operably linked to a therapeutic sequence comprises or is a lineage-specific promoter. In some embodiments, a promoter operably linked to a therapeutic sequence comprises or is a ubiquitous promoter. In some embodiments, a promoter operably linked to a therapeutic sequence comprises or is a CTSG-FES promoter. In some embodiments, a promoter operably linked to a therapeutic sequence comprises or is the nucleic acid sequence of any one of SEQ ID NOs: 88-106.

[0044] In some embodiments, a payload cassette comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of SEQ ID NO: 80. In some embodiments, a payload cassette comprises or is the nucleic acid sequence of SEQ ID NO: 80.

[0045] In some embodiments, a genome of a HDAd payload vector comprises a chromatin insulator. In some embodiments, a chromatin insulator comprises or is the nucleic acid sequence of SEQ ID NO: 133.

[0046] In some embodiments, a selection marker is MGMTP140K. In some embodiments, a nucleic acid sequence encoding a selection marker comprises or is the nucleic acid sequence of SEQ ID NO: 81. In some embodiments, a selection marker is a CD117 polypeptide comprising one or more mutations.

[0047] In some embodiments, a promoter operably linked to a nucleic acid sequence encoding a selection marker comprises or is a ubiquitous promoter. In some embodiments, a promoter operably linked to a nucleic acid sequence encoding a selection marker comprises or is an EF1a, ACTB, EFS, eIF4A1, CAG, CMV, GAPDH, KIN, PGK, ROSA, SFFV, SV40, UBB, or UBC promoter. In some embodiments, a promoter operably linked to a nucleic acid sequence encoding a selection marker comprises or is an EF1a promoter. In some embodiments, a promoter operably linked to a nucleic acid sequence encoding a selection marker comprises or is the nucleic acid sequence of any one of SEQ ID NOs: 107-127, 171, and 172.

[0048] In some embodiments, a selection cassette comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of SEQ ID NO: 83. In some embodiments, a selection cassette comprises or is the nucleic acid sequence of SEQ ID NO: 83.

[0049] In some embodiments, a first transposase target site is a Sleeping Beauty (SB) transposase target site and a second transposase target site is a SB transposase target site.

[0050] In some embodiments, a genome of a HDAd payload vector comprises a first recombinase target site and a second recombinase target site. In some embodiments, a genome of a HDAd payload vector comprises a first recombinase target site and a second recombinase target site, wherein a first transposase target site and a second transposase target site are located between the first recombinase target site and the second recombinase target site. In some embodiments, a genome of a HDAd payload vectorcomprises a first recombinase target site and a second recombinase target site, wherein a packaging sequence is located 5’ to the first recombinase target site. In some embodiments, a genome of a HDAd payload vector comprises a first recombinase target site and a second recombinase target site, wherein a first transposase target site and a second transposase target site are located between the first recombinase target site and the second recombinase target site and a packaging sequence is located 5’ to the first recombinase target site. In some embodiments, a first recombinase target site is a Flp recombinase target site and a second recombinase target site is a Flp recombinase target site. In some embodiments, a first recombinase target site is a Flpx9 or FlpE recombinase target site and a second recombinase target site is a Flpx9 or FlpE recombinase target site.

[0051] In some embodiments, a genome of a HDAd payload vector comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of SEQ ID NO: 134. In some embodiments, a genome of the HDAd payload vector comprises or is the nucleic acid sequence of SEQ ID NO: 134.

[0052] In some embodiments, a HDAd payload vector is for use in humans.

[0053] In another aspect, the present disclosure provides a cell comprising a HDAd payload vector, e.g., a HDAd payload provided herein. In some embodiments, a cell is a hematopoietic stem cell (HSC).

[0054] In another aspect, the present disclosure provides a pharmaceutical composition comprising a HDAd payload vector, e.g., a HDAd payload provided herein and one or more pharmaceutically acceptable carriers. In some embodiments, a pharmaceutical composition comprises a HDAd payload vector and a HDAd integration vector. In some embodiments, a pharmaceutical composition is a liquid composition. In some embodiments, a pharmaceutical composition comprises a buffer. In some embodiments, a pharmaceutical composition is formulated for injection to a subject in need thereof. In some embodiments, a pharmaceutical composition is formulated for use in humans.

[0055] In another aspect, the present disclosure provides a gene therapy system comprising a HDAd payload vector, e.g., a HDAd payload vector provided herein, and a HDAd integration vector, e.g., a HDAd integration vector provided herein. In some embodiments, a gene therapy system comprises (I) a HDAd payload vector comprising a CD46-binding adenoviral capsid and a double-stranded DNA genome comprising (1) a 5’ ITR and a 3’ ITR; (2) a packaging sequence; (3) a first transposase target site and a second transposase target site; (4) a payload cassette comprising (a) a therapeutic sequence comprising a nucleic acid sequence encoding a therapeutic polypeptide, wherein the therapeutic polypeptides is a CYBB, CYBA, NCF1, NCF2, NCF4, or CYBC1 therapeutic polypeptide; and (b) a promoter operably linked to the therapeutic sequence; and (5) a selection cassette comprising (a) a nucleic acid sequence encoding a selection marker; and (b) a promoter operably linked to the nucleic acid sequence encoding the selection marker; wherein (i) the payload cassette and the selection cassette are located between the first transposasetarget site and the second transposase target site; and (ii) the packaging sequence is located 5’ to the first transposase target site; and (II) a HDAd integration vector comprising a CD46-binding adenoviral capsid and a double-stranded DNA genome comprising (1) a 5’ ITR and a 3’ ITR; (2) a packaging sequence; (3) a nucleic acid sequence encoding a transposase.

[0056] In some embodiments, a CD46-binding adenoviral capsid of a HDAd payload vector is an Ad5 / 35 capsid. In some embodiments, a CD46-binding adenoviral capsid of a HDAd payload vector is an Ad5 / 35++ capsid. In some embodiments, a CD46-binding adenoviral capsid of a HDAd integration vector is an Ad5 / 35 capsid. In some embodiments, a CD46-binding adenoviral capsid of a HDAd integration vector is an Ad5 / 35++ capsid.

[0057] In some embodiments, a therapeutic sequence comprises or is a nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments, a therapeutic sequence comprises or is a nucleic acid sequence encoding a therapeutic nucleic acid. In some embodiments, a nucleic acid sequence encoding a therapeutic polypeptide is codon-optimized. In some embodiments, a therapeutic polypeptide comprises or is a CYBB therapeutic polypeptide. In some embodiments, a therapeutic polypeptide comprises or is a CYBA therapeutic polypeptide. In some embodiments, a therapeutic polypeptide comprises or is a NCF1 therapeutic polypeptide. In some embodiments, a therapeutic polypeptide comprises or is a NCF2 therapeutic polypeptide. In some embodiments, a therapeutic polypeptide comprises or is a NCF4 therapeutic polypeptide. In some embodiments, a therapeutic polypeptide comprises or is a CYBC1 therapeutic polypeptide. In some embodiments, a nucleic acid sequence encoding a therapeutic polypeptide comprises a nucleic acid sequence that has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the nucleic acid sequence of SEQ ID NO: 79. In some embodiments, a nucleic acid sequence encoding a therapeutic polypeptide comprises or is the nucleic acid sequence of SEQ ID NO: 79. In some embodiments, a therapeutic polypeptide comprises or is the amino acid sequence of any one of SEQ ID NOs: 151-161.

[0058] In some embodiments, a 5’ ITR of a genome of a HDAd payload vector is an Ad55’ ITR. In some embodiments, a 3’ ITR of a genome of a HDAd payload vector is an Ad5 3’ ITR. In some embodiments, a 5’ ITR of a genome of a HDAd payload vector is an Ad55’ ITR and a 3’ ITR of a genome of a HDAd payload vector is an Ad53’ ITR. In some embodiments, a packaging sequence of a genome of a HDAd payload vector is an Ad5 packaging sequence.

[0059] In some embodiments, a promoter operably linked to a therapeutic sequence comprises or is a lineage-specific promoter. In some embodiments, a promoter operably linked to a therapeutic sequence comprises or is a ubiquitous promoter. In some embodiments, a promoter operably linked to a therapeutic sequence comprises or is a CTSG-FES promoter. In some embodiments, a promoter operably linked to a therapeutic sequence comprises or is the nucleic acid sequence of any one of SEQ ID NOs: 88-106 and 175.

[0060] In some embodiments, a payload cassette comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of SEQ ID NO: 80. In some embodiments, a payload cassette comprises or is the nucleic acid sequence of SEQ ID NO: 80.

[0061] In some embodiments, a genome of a HDAd payload vector comprises a chromatin insulator. In some embodiments, a chromatin insulator comprises or is the nucleic acid sequence of SEQ ID NO: 133.

[0062] In some embodiments, a selection marker is MGMTP140K. In some embodiments, a nucleic acid sequence encoding a selection marker comprises or is the nucleic acid sequence of SEQ ID NO: 81. In some embodiments, a selection marker is a CD117 polypeptide comprising one or more mutations.

[0063] In some embodiments, a promoter operably linked to a nucleic acid sequence encoding a selection marker comprises or is a ubiquitous promoter. In some embodiments, a promoter operably linked to a nucleic acid sequence encoding a selection marker comprises or is an EF1a, ACTB, EFS, eIF4A1, CAG, CMV, GAPDH, KIN, PGK, ROSA, SFFV, SV40, UBB, or UBC promoter. In some embodiments, a promoter operably linked to a nucleic acid sequence encoding a selection marker comprises or is an EF1a promoter. In some embodiments, a promoter operably linked to a nucleic acid sequence encoding a selection marker comprises or is the nucleic acid sequence of any one of SEQ ID NOs: 107-127, 171, and 172.

[0064] In some embodiments, a selection cassette comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of SEQ ID NO: 83. In some embodiments, a selection cassette comprises or is the nucleic acid sequence of SEQ ID NO: 83.

[0065] In some embodiments, a first transposase target site is a Sleeping Beauty (SB) transposase target site and a second transposase target site is a SB transposase target site.

[0066] In some embodiments, a genome of a HDAd payload vector comprises a first recombinase target site and a second recombinase target site. In some embodiments, a genome of a HDAd payload vector comprises a first recombinase target site and a second recombinase target site, wherein a first transposase target site and a second transposase target site are located between the first recombinase target site and the second recombinase target site. In some embodiments, a genome of a HDAd payload vector comprises a first recombinase target site and a second recombinase target site, wherein a packaging sequence is located 5’ to the first recombinase target site. In some embodiments, a genome of a HDAd payload vector comprises a first recombinase target site and a second recombinase target site, wherein a first transposase target site and a second transposase target site are located between the first recombinase target site and the second recombinase target site and a packaging sequence is located 5’ to the first recombinase target site. In some embodiments, a first recombinase target site is a Flp recombinase targetsite and a second recombinase target site is a Flp recombinase target site. In some embodiments, a first recombinase target site is a Flpx9 or FlpE recombinase target site and a second recombinase target site is a Flpx9 or FlpE recombinase target site.

[0067] In some embodiments, a transposase comprises or is a Sleeping Beauty (SB) transposase. In some embodiments, a SB transposase is a SB10 transposase, SB100X transposase, SB100X+ transposase, or M3A transposase or a variant thereof. In some embodiments, a transposase comprises or is the amino acid sequence of SEQ ID NO: 135-138.

[0068] In some embodiments, a genome of an HDAd integration vector comprises a nucleic acid sequence encoding a recombinase. In some embodiments, a recombinase comprises or is a Flp recombinase. In some embodiments, a Flp recombinase comprises one or more mutations, optionally wherein one or more mutations are selected from P2S, L33S, Y108N, S294P, or a combination thereof. In some embodiments, a Flp recombinase comprises or is a Flpx9 recombinase, FlpE recombinase, or FlpO recombinase. In some embodiments, a recombinase comprises or is the amino acid sequence of any one of SEQ ID NOs: 139-141.

[0069] In some embodiments, a 5’ ITR of a genome of a HDAd integration vector is an Ad55’ ITR. In some embodiments, a 3’ ITR of a genome of a HDAd integration vector is an Ad53’ ITR. In some embodiments, a 5’ ITR of a genome of a HDAd integration vector is an Ad55’ ITR and a 3’ ITR of a genome of a HDAd integration vector is an Ad53’ ITR. In some embodiments, a packaging sequence of a genome of a HDAd integration vector is an Ad5 packaging sequence.

[0070] In some embodiments, a genome of a HDAd integration vector comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of any one of SEQ ID NOs: 144-146. In some embodiments, a genome of a HDAd integration vector comprises or is the nucleic acid sequence of any one of SEQ ID NOs: 144-146.

[0071] In some embodiments, a gene therapy system comprises or is an in vivo gene therapy system. In some embodiments, a gene therapy system is for use in humans.

[0072] In another aspect, the present disclosure provides a pharmaceutical composition comprising a HDAd payload vector and a HDAd integration vector, wherein the HDAd payload vector and HDAd integration vector are at a ratio of about 1-3:1 or 1:1-3 HDAd payload vector:HDAd integration vector, and wherein the HDAd payload vector comprises a CD46-binding adenoviral capsid and a double-stranded DNA genome comprising (1) a 5’ ITR and a 3’ ITR; (2) a packaging sequence; (3) a first recombinase target site and a second recombinase target site; (4) a first transposase target site and a second transposase target site; (5) a payload cassette comprising a nucleic acid sequence of SEQ ID NO: 80; and (6) a selection cassette comprising a nucleic acid sequence of SEQ ID NO: 83; wherein (i) the first transposase target siteand the second transposase target site are located between the first recombinase target site and the second recombinase target site; (ii) the payload cassette and the selection cassette are located between the first transposase target site and the second transposase target site; and (iii) the packaging sequence is located 5’ to the first recombinase target site; and wherein the HDAd integration vector comprises a CD46-binding adenoviral capsid and a double-stranded DNA genome comprising (1) a 5’ ITR and a 3’ ITR; (2) a packaging sequence; (3) a nucleic acid sequence encoding a recombinase; and (4) a nucleic acid sequence encoding a transposase.

[0073] In some embodiments, a CD46-binding adenoviral capsid of a HDAd payload vector is an Ad5 / 35 capsid. In some embodiments, a CD46-binding adenoviral capsid of a HDAd payload vector is an Ad5 / 35++ capsid. In some embodiments, a CD46-binding adenoviral capsid of a HDAd integration vector is an Ad5 / 35 capsid. In some embodiments, a CD46-binding adenoviral capsid of a HDAd integration vector is an Ad5 / 35++ capsid.

[0074] In some embodiments, a 5’ ITR of a genome of a HDAd payload vector is an Ad55’ ITR. In some embodiments, a 3’ ITR of a genome of a HDAd payload vector is an Ad5 3’ ITR. In some embodiments, a 5’ ITR of a genome of a HDAd payload vector is an Ad55’ ITR and a 3’ ITR of a genome of a HDAd payload vector is an Ad53’ ITR. In some embodiments, a packaging sequence of a genome of a HDAd payload vector is an Ad5 packaging sequence.

[0075] In some embodiments, a genome of a HDAd payload vector comprises a chromatin insulator. In some embodiments, a chromatin insulator comprises or is the nucleic acid sequence of SEQ ID NO: 133.

[0076] In some embodiments, a first transposase target site is a Sleeping Beauty (SB) transposase target site and a second transposase target site is a SB transposase target site.

[0077] In some embodiments, a first recombinase target site is a Flp recombinase target site and a second recombinase target site is a Flp recombinase target site. In some embodiments, a first recombinase target site is a Flpx9 or FlpE recombinase target site and a second recombinase target site is a Flpx9 or FlpE recombinase target site.

[0078] In some embodiments, a genome of a HDAd payload vector comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of SEQ ID NO: 134. In some embodiments, a genome of the HDAd payload vector comprises or is the nucleic acid sequence of SEQ ID NO: 134.

[0079] In some embodiments, a 5’ ITR of a genome of a HDAd integration vector is an Ad55’ ITR. In some embodiments, a 3’ ITR of a genome of a HDAd integration vector is an Ad53’ ITR. In some embodiments, a 5’ ITR of a genome of a HDAd integration vector is an Ad55’ ITR and a 3’ ITR of a genome of a HDAd integration vector is an Ad53’ ITR. In some embodiments, a packaging sequence of a genome of a HDAd integration vector is an Ad5 packaging sequence.

[0080] In some embodiments, a transposase comprises or is a Sleeping Beauty (SB) transposase. In some embodiments, a SB transposase is a SB10 transposase, SB100X transposase, SB100X+ transposase, or M3A transposase or a variant thereof. In some embodiments, a transposase comprises or is the amino acid sequence of SEQ ID NO: 135-138.

[0081] In some embodiments, a recombinase comprises or is a Flp recombinase. In some embodiments, a Flp recombinase comprises one or more mutations, optionally wherein one or more mutations are selected from P2S, L33S, Y108N, S294P, or a combination thereof. In some embodiments, a Flp recombinase comprises or is a Flpx9 recombinase, FlpE recombinase, or FlpO recombinase. In some embodiments, a recombinase comprises or is the amino acid sequence of any one of SEQ ID NOs: 139- 141.

[0082] In some embodiments, a genome of a HDAd integration vector comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of any one of SEQ ID NOs: 144-146. In some embodiments, a genome of a HDAd integration vector comprises or is the nucleic acid sequence of any one of SEQ ID NOs: 144-146. In some embodiments, a genome of a HDAd integration vector comprises or is the nucleic acid sequence of SEQ ID NO: 144. In some embodiments, a genome of a HDAd integration vector comprises or is the nucleic acid sequence of SEQ ID NO: 145. In some embodiments, a genome of a HDAd integration vector comprises or is the nucleic acid sequence of SEQ ID NO: 146.

[0083] In some embodiments, a pharmaceutical composition is a liquid composition. In some embodiments, a pharmaceutical composition comprises a buffer. In some embodiments, a pharmaceutical composition is formulated for injection to a subject in need thereof.

[0084] In another aspect, the present disclosure provides a HDAd integration vector, e.g., a HDAd integration vector provided herein. In some embodiments, a HDAd integration vector comprises a CD46- binding adenoviral capsid and a double-stranded DNA genome comprising (1) a 5’ ITR and a 3’ ITR; (2) a packaging sequence; (3) nucleic acid sequence encoding a Flp recombinase comprising the amino acid sequence of SEQ ID NO: 140; (4) a promoter operably linked to the nucleic acid sequence encoding a Flp recombinase and comprising SEQ ID NO: 172; (5) a nucleic acid sequence encoding a Sleeping Beauty (SB) transposase comprising the amino acid sequence of SEQ ID NO: 136; and (6) a promoter operably linked to the nucleic acid sequence encoding a SB transposase and comprising SEQ ID NO: 171. In some embodiments, a HDAd integration vector comprises a CD46-binding adenoviral capsid and a double- stranded DNA genome comprising (1) a 5’ ITR and a 3’ ITR; (2) a packaging sequence; (3) nucleic acid sequence encoding a Flp recombinase comprising the amino acid sequence of SEQ ID NO: 140; (4) a promoter operably linked to the nucleic acid sequence encoding a Flp recombinase and comprising SEQ ID NO: 171; (5) a nucleic acid sequence encoding a Sleeping Beauty (SB) transposase comprising theamino acid sequence of SEQ ID NO: 136; and (6) a promoter operably linked to the nucleic acid sequence encoding a SB transposase and comprising SEQ ID NO: 172.

[0085] In some embodiments, a CD46-binding adenoviral capsid of a HDAd integration vector is an Ad5 / 35 capsid. In some embodiments, a CD46-binding adenoviral capsid of a HDAd integration vector is an Ad5 / 35++ capsid.

[0086] In some embodiments, a 5’ ITR of a genome of a HDAd integration vector is an Ad55’ ITR. In some embodiments, a 3’ ITR of a genome of a HDAd integration vector is an Ad53’ ITR. In some embodiments, a 5’ ITR of a genome of a HDAd integration vector is an Ad55’ ITR and a 3’ ITR of a genome of a HDAd integration vector is an Ad53’ ITR. In some embodiments, a packaging sequence of a genome of a HDAd integration vector is an Ad5 packaging sequence.

[0087] In some embodiments, a genome of a HDAd integration vector comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of SEQ ID NO: 145. In some embodiments, a genome of a HDAd integration vector comprises or is the nucleic acid sequence of SEQ ID NO: 145. In some embodiments, a genome of a HDAd integration vector comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of SEQ ID NO: 146. In some embodiments, a genome of a HDAd integration vector comprises or is the nucleic acid sequence of SEQ ID NO: 146.

[0088] In some embodiments, a HDAd integration vector is for use in humans. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1. Schematic of an exemplary HDAd5 / 35++-CYBB vector genome. ITR = inverted terminal repeat; Ad5 Ψ = human adenovirus serotype 5 packaging sequence; MAMLD1 = mastermind like domain-1; FRT = flippase recombination target site; SBpT4 RIR = optimized (pT4) sleeping beauty inverted repeat; hEFIAp = human elongation factor-1 alpha promoter; hMGMT[P140K] = human methylguanine methyltransferase (MGMT) comprising a P140K mutation; SV40pA = simian virus-40 polyadenylation signal; cHS4 = chicken hypersensitive site 4 insulator; bGHpA = bovine growth hormone polyadenylation signal; WPRE mut6delATG = woodchuck hepatitis virus post-transcriptional regulatory element, mutated to exclude promoter (mut6) and start codons (delATG) of endogenous woodchuck hepatitis virus protein X; coCYBB = codon optimized CYBB; CTSG-FESp = Cathepsin G / c-fes chimeric promoter.

[0090] Figure 2. Schematic of an exemplary HDAd5 / 35++-SB / Flp vector genome. ITR = inverted terminal repeat; MAMLD1 = mastermind like domain-1; hEFIAp = human elongation factor-1 alpha promoter; FLP = flippase; FLP 3’UTR = FLP 3’ untranslated region; hBGpA = human beta-globinpolyadenylation signal; bGHpA = bovine growth hormone polyadenylation signal; SB = Sleeping Beauty transposase; SV40 intron = simian virus-40 intron; mPGKp = murine phosphoglycerate kinase promoter; Ad5 Ψ = human adenovirus serotype 5 packaging sequence.

[0091] Figure 3. Diagram of in vivo gene therapy using adenoviral vectors as described herein.

[0092] Figure 4. HDAd5 / 35++-CYBB can provide high levels of transduction of cells and expression of CYBB in cells in vitro. In brief, wild-type THP-1 cells (“WT”) or THP-1 cells comprising CYBB knockout (“CYBB KO”) were transduced on day 0 with HDAd5 / 35++-CYBB for 24 hours. Cells were washed after 24 hours and cultured for 4 additional days. Untreated control cells were also assessed. (A) On day 4, flow cytometry analysis of MGMT expression was used to determine the percentage of cells that were transduced. (B) Further flow cytometry analysis of CYBB expression was used to determine the percentage of transduced (MGMT+) cells that expressed CYBB.

[0093] Figure 5. HDAd5 / 35++-CYBB + HDAd5 / 35++-SB / Flp can provide increased CYBB activity in cells ex vivo. (A) Exemplary schematic of transduction and differentiation of cells. In brief, CD46tg / CYBB KO lineage negative (Lin-) cells were transduced with HDAd5 / 35++-CYBB and HDAd5 / 35++-SB / Flp (each at a MOI of 2000) for 3 days. Cells were maintained in non-differentiation media containing Flt3L, IL-11, IL-3, and stem cell factor (SCF). After 3 days, Lin- cells were cultured in media containing IL-3 and SCF for 2 days, cultured in media containing G-CSF, IL-3, and SCF for an additional 2 days, and then cultured in media containing G-CSF for an additional 3 or 4 days to finally primarily yield terminally differentiated polymorphonuclear neutrophils. (B) On day 12, resulting neutrophils were assessed using a DHR assay as described herein to determine CYBB activity, as reflected in NADPH oxidase activity. Flow cytometry, using live / dead dye and various antibodies (e.g., anti-CD45, anti-CD11b, anti-Ly6g, anti-CYBB antibodies), was used to measure the percentage of neutrophils that were positive for DHR activity. Cells that were not transduced with HDAd5 / 35++-CYBB and HDAd5 / 35++-SB / Flp (“Untreated”) were also examined. Exemplary DHR assay results are displayed in the graph.

[0094] Figure 6. HDAd5 / 35++ can provide high levels of transduction of human primitive HSC populations. In brief, plerixafor-mobilized CD34+ cells collected from two human donors (“Donor 1” and “Donor 2”) were transduced with HDAd5 / 35++ vector containing a GFP reporter sequence at a MOI of 300, 1000, or 3000 for 80 hours. The HDAd5 / 35++ vector containing a GFP reporter sequence is similar to the HDAd5 / 35++-CYBB but contains a GFP reporter sequence in place of a CYBB sequence. Levels of transduction and expression were assessed by measuring percentage of cells that express GFP by flow cytometry. Exemplary results are shown in the graphs. The left graphs depict percentage of GFP+ cells as measured by flow cytometry. The right graphs depict geometric mean fluorescence intensity (MFI). For each MOI (300, 1000, 3000) on each graph, the left column represents data from CD34+ high cells and theright column represents data from CD34+ CD90+ CD45RA- cells.

[0095] Figure 7. HDAd5 / 35++ transduction can be affected by level of expression of CD46 on red blood cells (RBCs). In brief, whole blood from human, CD46tg mice, C57BL / 6 mice, rhesus monkey, and cynomolgus monkey was washed twice with PBS. Resulting washed whole blood was then added to HEK293 cells and followed by addition of HDAd5 / 35++ vector containing a GFP reporter sequence at a MOI of 1, 10, 100, 500, or 1000. After incubation for 1 hour, cells were washed twice with PBS and incubated for an additional 48 hours. Cells were then assessed for expression of GFP using flow cytometry. Exemplary data are shown in graph. The X-axis displays the MOI tested; the Y-axis depicts percentage of GFP+ cells. For each MOI (no virus, 1, 10, 100, 500, 1000), the columns represent from left to right: No Blood, C57BL / 6, Human, CD46tg, Rhesus, Cynomolgus.

[0096] Figure 8. Exemplary schematic of an in vivo mouse study of HDAd5 / 35++ transduction. In brief, female (F) CD46tg mice (n=5 per group) were subcutaneously administered G-CSF (250 ug / kg) on days -4 to -1 and plerixafor (5 mg / kg) on day 0 at -40 minutes for mobilization of HSCs. Mice received an immunosuppression regimen comprising intraperitoneally administered dexamethasone (10 mg / kg) at 15 hours and 2 hours prior to administration of vector. HDAd5 / 35++ GFP and HDAd5 / 35++-SB / Flp were intravenously administered at a 1:1 ratio at 1.5 x 1012GC / kg, 3.0 x 1012GC / kg, 6.25 x 1012GC / kg, or 1.2 x 1013GC / kg to the mice. After 4 weeks, mice were administered an enrichment regimen comprising O6BG (30 mg / kg) and an increasing dose of BCNU (5 mg / kg, 5 mg / kg, 7.5 mg / kg, and 10 mg / kg) administered intraperitoneally for 4 rounds, 2 weeks apart. A group of mice that received vehicle instead of vector and an untreated group of mice that did not receive the vector or mobilization were also examined. A subset of mice dosed with either 6.25 x 1012GC / kg or 1.2 x 1013GC / kg of the vectors were sacrificed at day 7 post- vector administration and tissues collected. Remaining mice were sacrificed at week 20 and tissues collected.

[0097] Figure 9. HDAd5 / 35++ can provide high levels of integration into HSCs and gene marking following enrichment with O6BG / BCNU. (A) GFP expression in LSK cells (CD45+ Lin- Sca1+ c-Kit+) from bone marrow collected from mice in a study schematized in Figure 8 was assessed by flow cytometry. Exemplary data are shown in graphs. The X-axis displays treatment group (1.25E13 = 1.2 x 1013GC / kg; 6.25E12 = 6.25 x 1012GC / kg; 3.0E12 = 3.0 x 1012GC / kg; 1.5E12 = 1.5 x 1012GC / kg; Veh No Sel = vehicle without enrichment regimen; No SB 6.25E12 = 6.25 x 1012GC / kg of HDAd5 / 35++ GFP only; No Sel 6.25E12 = 6.25 x 1012GC / kg without enrichment regimen; Veh Sel = vehicle with enrichment regimen; UT = untreated); the Y-axis depicts percentage of GFP+ LSK cells. Data from individual samples are shown as circles. Means are represented by horizontal line. (B) GFP expression in LSK cells (CD45+ Lin- Sca1+ c-Kit+) from spleens collected from mice in a study schematized in Figure 8 was assessed by flow cytometry. Exemplary data are shown in graphs. The X-axis displays treatment group (1.25E13 = 1.2 x1013GC / kg; 6.25E12 = 6.25 x 1012GC / kg; 3.0E12 = 3.0 x 1012GC / kg; 1.5E12 = 1.5 x 1012GC / kg; Veh No Sel = vehicle without enrichment regimen; No SB 6.25E12 = 6.25 x 1012GC / kg of HDAd5 / 35++ GFP only; No Sel 6.25E12 = 6.25 x 1012GC / kg without enrichment regimen; Veh Sel = vehicle with enrichment regimen; UT = untreated); the Y-axis depicts percentage of GFP+ LSK cells. Data from individual samples are shown as circles. Means are represented by horizontal line.

[0098] Figure 10. Exemplary schematic of an in vivo mouse study of transduction with HDAd5 / 35++-CYBB + HDAd5 / 35++-SB / Flp. In brief, male (M) CD46tg mice (n=5 per group) were subcutaneously administered G-CSF (250 ug / kg) on days -4 to -1 and plerixafor (5 mg / kg) on day 0 at -40 minutes for mobilization of HSCs. Mice received an immunosuppression regimen comprising intraperitoneally administered dexamethasone (10 mg / kg) at 15 hours and 2 hours prior to administration of vector. HDAd5 / 35++-CYBB and HDAd5 / 35++-SB / Flp were intravenously administered at a 1:1 ratio at 6.25 x 1012GC / kg or 1.2 x 1013GC / kg to the mice. For 4 weeks following administration of HDAd5 / 35++-CYBB and HDAd5 / 35++-SB / Flp, mice received a chronic immunosuppression regimen comprising mycophenolate mofetil (20 mg / kg), methylprednisolone succinate (20 mg / kg), and rapamycin (0.2 mg / kg) intraperitoneally administered 3 times a week. After 4 weeks, mice were administered an enrichment regimen comprising O6BG (30 mg / kg) and an increasing dose of BCNU (5 mg / kg, 5 mg / kg, 7.5 mg / kg, and 10 mg / kg) administered intraperitoneally for 4 rounds, 2 weeks apart. Following the last round of the enrichment regimen, mice were again administered the chronic immunosuppression regimen comprising mycophenolate mofetil (20 mg / kg), methylprednisolone succinate (20 mg / kg), and rapamycin (0.2 mg / kg) intraperitoneally administered 3 times a week for 10 additional weeks. An untreated group of mice that did not receive any of HDAd5 / 35++-CYBB and HDAd5 / 35++-SB / Flp, mobilization regimen (G- CSF and plerixafor), immunosuppression regimen (dexamethasone or chronic), and enrichment regimen (O6BG and BCNU) were also examined. Mice were sacrificed at week 20 and tissues collected. White blood cells (WBCs) were collected at various time points during the study.

[0099] Figure 11. HDAd5 / 35++-CYBB + HDAd5 / 35++-SB / Flp can provide high levels of CYBB payload integration in vivo. WBCs collected from mice at various time points (11.5 weeks, 13.5 weeks, 15.5 weeks) in a study schematized in Figure 10 were analyzed using qPCR to determine integration of CYBB payload. Exemplary data are shown in the graph. The X-axis depicts study week when WBCs were collected; the Y-axis depicts vector copy number (VCN) per cell. Error bars represent standard deviation.

[0100] Figure 12. HDAd5 / 35++-CYBB + HDAd5 / 35++-SB / Flp can provide high levels of CYBB payload integration in neutrophils, spleen cells, and total bone marrow cells in vivo. Tissues collected following sacrifice of mice in a study schematized in Figure 10 were analyzed using qPCR to determine integration of CYBB payload. The X-axis displays assessed cell type; the Y-axis depicts vector copy number (VCN) per cell. Mean VCN per cell values (e.g., “0.42”) are shown above each column. Datafrom individual samples are shown as black shapes (triangles, squares). Error bars represent standard deviation. Treatment groups are shown at bottom.

[0101] Figure 13. HDAd5 / 35++-CYBB + HDAd5 / 35++-SB / Flp can provide high levels of CYBB payload expression in neutrophils in vivo. Neutrophils collected from mouse tissues collected following sacrifice at week 20 in a study schematized in Figure 10 were analyzed using RT-qPCR to determine level of expression of CYBB mRNA relative to expression of ACTB mRNA. The X-axis displays treatment group; the Y-axis depicts log fold expression of CYBB relative to ACTB as normalized to the untreated group. Data from individual samples are shown as black shapes (circles, triangles, squares). Error bars represent standard deviation.

[0102] Figure 14. Mobilization of HSCs in CD46tg mice and CD46tg / CYBB KO mice is comparable. In brief, male CD46tg mice or male CD46tg / CYBB KO mice (n=3 per group) were subcutaneously administered a mobilization regimen comprising G-CSF (250 ug / kg) on days -4 to 0 and plerixafor (5 mg / kg) at -40 minutes. Mice were also intraperitoneally administered dexamethasone (10 mg / kg) at -15 hours and -2 hours. Untreated groups of both male CD46tg and male CD46tg / CYBB KO mice were also examined. Blood was collected on day 1 via a submandibular bleed. (A) Blood samples were subjected to a colony forming unit (CFU) assay. Exemplary results are displayed in the graph. The X-axis displays treatment group (Mob = mice receiving mobilization regimen and dexamethasone; UT = untreated mice); the Y-axis depicts average colonies per well. Data from individual mice are shown as shapes (filled circles, unfilled circles). Error bars represent standard deviation. (B) Flow cytometry was used to measure progenitor cell (Lin- c-Kit+) population. Exemplary results are displayed in the graph. The X-axis displays treatment group (Mob: mice receiving mobilization regimen and dexamethasone; UT: untreated mice); the Y-axis depicts frequency of Lin- c-Kit+ cells among living single cells. Data from individual mice are shown as shapes (filled circles, unfilled circles). Error bars represent standard deviation. (C) Flow cytometry was used to measure LSK cell (CD45+ Lin- Sca1+ c-Kit+) population. Exemplary results are displayed in the graph. The X-axis displays treatment group (Mob: mice receiving mobilization regimen and dexamethasone; UT: untreated mice); the Y-axis depicts frequency of CD45+ Lin- Sca1+ c-Kit+ cells among living single cells. Data from individual samples are shown as shapes (filled circles, unfilled circles). Error bars represent standard deviation.

[0103] Figure 15. Exemplary schematic of an in vivo mouse study of transduction with HDAd5 / 35++-CYBB + HDAd5 / 35++-SB / Flp. In brief, male (M) CD46tg / CYBB KO mice were intraperitoneally administered G-CSF (250 ug / kg) on days -4 to -2, subcutaneously administered G-CSF (250 ug / kg) on day -1, and subcutaneously administered plerixafor (5 mg / kg) on day 0 for mobilization of HSCs. Mice received an immunosuppression regimen comprising intraperitoneally administered dexamethasone (10 mg / kg) at 15 hours and 2 hours prior to administration of vector. HDAd5 / 35++-CYBBand HDAd5 / 35++-SB / Flp were intravenously administered at a 1:1 ratio at 6.25 x 1012GC / kg to the mice (n=8). For 4 weeks following administration of HDAd5 / 35++-CYBB and HDAd5 / 35++-SB / Flp, mice received an immunosuppression regimen comprising mycophenolate mofetil (20 mg / kg), methylprednisolone succinate (20 mg / kg), and rapamycin (0.2 mg / kg) intraperitoneally administered 3 times a week. After 4 weeks, mice were administered an enrichment regimen comprising O6BG (30 mg / kg) and an increasing dose of BCNU (5 mg / kg, 5 mg / kg, 7.5 mg / kg, and 10 mg / kg) administered intraperitoneally for 4 rounds, 2 weeks apart. Following the last round of the enrichment regimen, mice were again administered the immunosuppression regimen comprising mycophenolate mofetil (20 mg / kg), methylprednisolone succinate (20 mg / kg), and rapamycin (0.2 mg / kg) intraperitoneally administered 3 times a week for 8 additional weeks. A group of mice (n=3) that received the mobilization regimen and saline only were also examined. Mice were sacrificed at week 18 and tissues collected. White blood cells (WBCs) were collected at various time points during the study.

[0104] Figure 16. HDAd5 / 35++-CYBB + HDAd5 / 35++-SB / Flp can provide increased levels of CYBB protein and activity in neutrophils in vivo. (A) Neutrophils (CD45+ CD11b+ Ly6G+) from WBCs collected from mice at various time points (12 weeks, 15 weeks, 18 weeks) in a study schematized in Figure 15 were analyzed for CYBB protein expression using flow cytometry. Exemplary data are shown in the graph. Treatment groups are shown at top. The X-axis depicts study week when WBCs were collected; the Y-axis depicts percentage of CYBB+ neutrophils. Data from individual samples are shown as shapes (circles, squares). Error bars represent standard deviation. (B) Neutrophils (CD45+ CD11b+ Ly6G+) from WBCs collected from mice at various time points (12 weeks, 15 weeks, 18 weeks) in a study schematized in Figure 15 were analyzed for CYBB activity, as reflected in NADPH oxidase activity, using a DHR assay as described herein. Exemplary data are shown in the graph. Treatment groups are shown at top. The X- axis depicts study week when WBCs were collected; the Y-axis depicts percentage of DHR+ neutrophils. Data from individual samples are shown as shapes (circles, squares). Error bars represent standard deviation.

[0105] Figure 17. HDAd5 / 35++-CYBB + HDAd5 / 35++-SB / Flp can provide high levels of MGMT expression that correlate with high levels of CYBB payload integration in vivo. Tissues collected following sacrifice of mice in a study schematized in Figure 15 were analyzed using flow cytometry to determine levels of MGMT expression and using qPCR levels of CYBB payload integration. (A) MGMT expression in bone marrow (BM) lineage negative (Lin-) cells was assessed. Exemplary data are shown in graph. The X-axis displays treatment group (UT = untreated); the Y-axis depicts percentage of MGMT+ cells. Data from individual samples are shown as shapes (circles, squares). Error bars represent standard deviation. (B) MGMT expression in LSK cells from bone marrow (BM) was assessed. Exemplary data are shown in graph. The X-axis displays treatment group (UT = untreated); the Y-axis depicts percentage of MGMT+cells. Data from individual samples are shown as shapes (circles, squares). Error bars represent standard deviation. (C) MGMT expression in neutrophils (CD11b+ Ly6G+) from blood was assessed. Exemplary data are shown in graph. The X-axis displays treatment group (UT = untreated); the Y-axis depicts percentage of MGMT+ cells. Data from individual samples are shown as shapes (circles, squares). Error bars represent standard deviation. (D) CYBB payload integration in total bone marrow cells was assessed. Exemplary data are shown in graph. The X-axis displays mouse from HDAd5 / 35++-CYBB and HDAd5 / 35++-SB / Flp treatment group; the Y-axis depicts vector copy number (VCN) per cell.

[0106] Figure 18. Exemplary schematic of an in vivo mouse study of transduction with HDAd5 / 35++-CYBB + HDAd5 / 35++-SB / Flp. In brief, male (M) and female (F) CD46tg mice and male and female CD46tg / CYBB KO mice (n=4 male and 4 female) were subcutaneously administered G-CSF (250 ug / kg) on days -4 to -1 and plerixafor (5 mg / kg) on day 0 for mobilization of HSCs. Mice received an immunosuppression regimen comprising intraperitoneally administered dexamethasone (10 mg / kg) at 15 hours and 2 hours prior to administration of vector. HDAd5 / 35++-CYBB and HDAd5 / 35++-SB / Flp were intravenously administered at a 1:1 ratio at 6.25 x 1012GC / kg to the mice. For 4 weeks following administration of HDAd5 / 35++-CYBB and HDAd5 / 35++-SB / Flp, mice received an immunosuppression regimen comprising mycophenolate mofetil (20 mg / kg), methylprednisolone succinate (20 mg / kg), and rapamycin (0.2 mg / kg) intraperitoneally administered 3 times a week. After 4 weeks, mice were administered an enrichment regimen comprising O6BG (30 mg / kg) administered intraperitoneally and an increasing dose of TMZ (150 mg / kg) administered orally for 3 rounds, 4 weeks apart. A group of mice that received vehicle instead of HDAd5 / 35++-CYBB and HDAd5 / 35++-SB / Flp and did not receive the immunosuppression regimen and a group of mice that received the mobilization regimen only were also examined. Mice were sacrificed at week 19 and tissues collected. White blood cells (WBCs) were collected at various time points during the study.

[0107] Figure 19. HDAd5 / 35++-CYBB + HDAd5 / 35++-SB / Flp can provide high levels of CYBB protein and activity in neutrophils in vivo. (A) Neutrophils (CD45+ CD11b+ Ly6G+) from WBCs collected from CD46tg / CYBB KO mice at various time points in a study schematized in Figure 18 were analyzed for CYBB protein expression using flow cytometry. Exemplary data are shown in the graph. The X-axis depicts study week when WBCs were collected; the Y-axis depicts percentage of CYBB+ neutrophils. Dashed vertical lines represent weeks when mice received enrichment regimen (O6BG / TMZ). Error bars represent standard error of the mean (SEM). (B) Neutrophils (CD45+ CD11b+ Ly6G+) from WBCs collected from CD46tg / CYBB KO mice at various time points in a study schematized in Figure 18 were analyzed for CYBB activity, as reflected in NADPH oxidase activity, using a DHR assay as described herein. Exemplary data are shown in the graph. Treatment groups are shown at top. The X-axis depicts study week when WBCs were collected; the Y-axis depicts percentage of DHR+ neutrophils. Dashedvertical lines represent weeks when mice received enrichment regimen (O6BG / TMZ). Error bars represent SEM.

[0108] Figure 20. HDAd5 / 35++-CYBB + HDAd5 / 35++-SB / Flp can provide high levels of CYBB protein and activity in neutrophils in vivo. (A) Neutrophils (CD45+ CD11b+ Ly6G+) from WBCs collected from CD46tg / CYBB KO mice at week 19 in a study schematized in Figure 18 were analyzed for CYBB protein expression using flow cytometry. Exemplary data are shown in the graph. The X-axis displays treatment group (Mobilized Only = CD46 WT mice receiving only mobilization regimen; WT = CD46 WT control mice receiving not receiving any of HDAds, mobilization regimen, immunosuppression regimen, and enrichment regimen); the Y-axis depicts percentage of CYBB+ neutrophils. Data from individual samples are shown as shapes (circles, squares, diamonds, stars). Dashed horizontal line represents therapeutic threshold of 10%. Error bars represent SEM. (B) Neutrophils (CD45+ CD11b+ Ly6G+) from WBCs collected from CD46tg / CYBB KO mice at week 19 in a study schematized in Figure 18 were analyzed for CYBB activity, as reflected in NADPH oxidase activity, using a DHR assay as described herein. Exemplary data are shown in the graph. Treatment groups are shown at top. The X-axis displays treatment group (Mobilized Only = CD46 WT mice receiving only mobilization regimen; WT = CD46 WT control mice not receiving any of HDAds, mobilization regimen, immunosuppression regimen, and enrichment regimen); the Y-axis depicts percentage of DHR+ neutrophils. Data from individual samples are shown as shapes (circles, squares, diamonds, stars). Dashed horizontal line represents therapeutic threshold of 10%. Error bars represent SEM. (C) Neutrophils (CD45+ CD11b+ Ly6G+) from WBCs collected from CD46tg / CYBB KO mice at week 19 in a study schematized in Figure 18 were analyzed for MGMT protein expression using flow cytometry. Exemplary data are shown in the graph. The X-axis displays treatment group (KO-Mobilized Only = CD46 / CYBB KO mice receiving only the mobilization regimen; WT-Mobilized Only = CD46 WT mice receiving only the mobilization regimen); the Y-axis depicts percentage of CYBB+ neutrophils. Data from individual samples are shown as shapes (circles, squares, diamonds, stars). Dashed horizontal line represents therapeutic threshold of 10%. Error bars represent SEM.

[0109] Figure 21. Graph showing Sleeping beauty protein expression at day 2 in human T cells transduced with Integration Vector A (payload vector + integration vector comprising PGK-Sleeping Beauty / EF1a-FLP), Integration Vector B (payload vector + integration vector comprising EF1a-Sleeping Beauty / CAG-FLP), Integration Vector C (payload vector + integration vector comprising CAG-Sleeping Beauty / EF1a-FLP), or payload vector only (PL). Data are representative of three independent experiments.

[0110] Figure 22. Graph showing gene marking frequency at day 19 post-administration of HDAd in human T cells transduced with Integration Vector A (PGK-Sleeping Beauty / EF1a-FLP), Integration Vector B (EF1a-Sleeping Beauty / CAG-FLP), and Integration Vector C (CAG-Sleeping Beauty / EF1a-FLP) pairingwith the indicated payloads. Data are representative of two independent experiments.

[0111] Figure 23. Timeline of in vivo HDAd-mediated PBMC transduction to assess integration in human T cells in IL-15tg NSG mice.

[0112] Figure 24. Graph showing CD19 CAR expression in human T cells over time in the peripheral blood in IL15tg NSG mice. Integration Vector A (payload vector + integration vector comprising PGK- Sleeping Beauty / EF1a-FLP) N=5, Integration Vector B (payload vector + integration vector comprising EF1a-Sleeping Beauty / CAG-FLP) N=6, Integration Vector C (payload vector + integration vector comprising CAG-Sleeping Beauty / EF1a-FLP) N= 6.

[0113] Figure 25. Timeline of HDAd mediated transduction of HSPCs and neutrophil differentiation assay to assess integration in mouse HSPCs in vitro. Neutrophil differentiation was initiated at Day 3.

[0114] Figure 26. Graph showing frequency of GFP+ cells among live cells at day 3 and day 10 post- transduction. Integration Vector A (PGK-Sleeping Beauty / EF1a-FLP), Integration Vector B (EF1a- Sleeping Beauty / CAG-FLP), Integration Vector C (CAG-Sleeping Beauty / EF1a-FLP).

[0115] Figure 27. Timeline of in vivo HDAd-mediated HSC transduction and single cell CFU assay to assess integration in HSPCs.

[0116] Figure 28. Graph showing frequency of GFP+ cells in LSK cells derived from bone marrow of human CD46 transgenic (hCD46tg) mice at day 7 after injection with GFP-containing payload vector paired with different integration vectors. Integration Vector A (payload vector + integration vector comprising PGK-Sleeping Beauty / EF1a-FLP), Integration Vector B (payload vector + integration vector comprising EF1a-Sleeping Beauty / CAG-FLP), Integration Vector C (payload vector + integration vector comprising CAG-Sleeping Beauty / EF1a-FLP). Data represent mean + / - SEM. N=5 per group, except for N=3 for Mock.

[0117] Figure 29. Graph showing integration in mouse HSPCs transduced in vivo with GFP- containing payload vector paired with different integration vectors as measured by frequency of GFP+ CFUs among total CFUs.

[0118] Figure 30. Graph showing integration in human CD34+ cells as measured by frequency of GFP+ CFUs among total CFUs. Integration Vector A (payload vector + integration vector comprising PGK- Sleeping Beauty / EF1a-FLP) N=3, Integration Vector B (payload vector + integration vector comprising EF1a-Sleeping Beauty / CAG-FLP) N=3, Payload Vector Only (Payload only) N=2, Untreated (UT) N=2.

[0119] Figure 31. Timeline of in vivo HDAd-mediated HSC transduction and enrichment in mobilized human CD46 transgenic (hCD46tg) mice.

[0120] Figure 32. Graph showing frequency of payload expression in CD45+ cells in peripheral blood (PB). PB was collected 8 weeks after administration of HDAd vectors and 4 weeks after O6BG / TMZ administration. Untreated (UT) N=5, Integration Vector A (payload vector + integration vector comprisingPGK-Sleeping Beauty / EF1a-FLP) N=5, Integration Vector B (payload vector + integration vector comprising EF1a-Sleeping Beauty / CAG-FLP) N=5, Integration Vector A (payload vector + integration vector comprising PGK-Sleeping Beauty / EF1a-FLP) + O6BG / TMZ N=12, Integration Vector B (payload vector + integration vector comprising EF1a-Sleeping Beauty / CAG-FLP) + O6BG / TMZ N=22.

[0121] Figure 33. Graph showing frequency of payload expression in peripheral blood (PB) immune cells. PB was collected 8 weeks after administration of HDAd vectors and 4 weeks after O6BG / TMZ administration. Integration Vector A (payload vector + integration vector comprising PGK-Sleeping Beauty / EF1a-FLP) + O6BG / TMZ N=12, Integration Vector B (payload vector + integration vector comprising EF1a-Sleeping Beauty / CAG-FLP) + O6BG / TMZ N=22.

[0122] Figure 34. HDAd5 / 35++-CYBB + HDAd5 / 35++-SB / Flp can provide high levels of CYBB protein and activity in neutrophils in vivo. (A) Blood samples collected from CD46tg / CYBB KO mice at various time points in a study schematized in Figure 18 were analyzed for counts of circulating neutrophils. Exemplary data are shown in the graph. The X-axis depicts study week; the Y-axis depicts counts of circulating neutrophils as thousand (K) / ul of blood sample. Dashed vertical lines represent weeks when mice received enrichment regimen (O6BG / TMZ). Error bars represent standard error of the mean (SEM). (B) Bone marrow samples from CD46tg / CYBB KO mice at end of a study schematized in Figure 18 were assessed for frequency of neutrophils (CD11b+ Ly6G+) using flow cytometry. Exemplary data are shown in the graph. The X-axis displays treatment group; the Y-axis depicts frequency of CD11b+ Ly6G+ cells as a percentage. Error bars represent SEM. (C) CYBB payload integration in total bone marrow cells from CD46tg / CYBB KO mice at end of a study schematized in Figure 18 was assessed. Exemplary data are shown in graph. The X-axis displays treatment group; the Y-axis depicts vector copy number (VCN) per cell. Error bars represent SEM.

[0123] Figure 35. HDAd5 / 35++-CYBB + HDAd5 / 35++-SB / Flp can provide robust levels of MGMT expression, increased levels of proliferation- and / or differentiation-capable bone marrow cells, and increased levels of NADPH oxidase complex activity in circulating neutrophils in vivo. (A) Bone marrow (BM) samples from CD46tg / CYBB KO mice at end of a study schematized in Figure 18 were assessed for MGMT expression in lineage negative (Lin-) cells using flow cytometry. The X-axis displays treatment group; the Y-axis depicts percentage of MGMT+ cells. Error bars represent SEM. (B) Lineage negative cells isolated from bone marrow samples of CD46tg / CYBB KO mice at end of a study schematized in Figure 18 were assessed for colony formation. The X-axis displays treatment group; the Y-axis displays colony forming units (CFU) counts. Error bars represent SEM. (C) Circulating neutrophils collected from CD46tg / CYBB KO mice at week 19 in a study schematized in Figure 18 were analyzed using a flow- cytometry-based DHR assay as described herein. The X-axis displays treatment group; the Y-axis depicts mean fluorescent intensity (MFI) in DHR+ neutrophils. Error bars represent SEM.

[0124] Figure 36. Graph showing CD46 expression in various cell lineages in blood collected from CD46tg mice mobilized with G-CSF. The X-axis displays cell lineage type; the Y-axis displays CD46 geometric mean fluorescent intensity (gMFI) as measured by flow cytometry. LSK = Lin- Sca-1+ c-Kit+ HSCs.

[0125] Figure 37. Graphs showing serum chemistry analysis performed for alanine aminotransferase (ALT) in (A), aspartate aminotransferase (AST) in (B), total bilirubin in (C), C-reactive protein (CRP) in (D), and creatinine kinase (CK) in (E). Arrow indicates time of HDAd administration. NHP #1 was dosed with vehicle only, NHP #2 and #3 were dosed with HDAd5 / 35++ GFP and HDAd5 / 35++-SB / Flp at a 1:1 ratio at 6.25 x 1012GC / kg, and NHP #4, #5, and #6 were dosed with HDAd5 / 35++ GFP and HDAd5 / 35++- SB / Flp at a 1:1 ratio at 9.25 x 1012GC / kg. The reference range for ALT, AST, CK, and total bilirubin are as follows: ALT 25-82 (Male) / 18-79 (Female) U / L, AST 28-82 (Male) / 23-65 (Female) U / L, CK 96-773 (Male) / 82-672 (Female) U / L, and total bilirubin 0.1-0.3 (Male) / 0.1-0.4 (Female) mg / dL. Data from one animal removed due to baseline abnormality.

[0126] Figure 38. Graph showing hematology analysis performed for platelets (PLT). NHP #1 (Female) was dosed with vehicle only, NHP #2 (Male) and #3 (Female) were dosed with HDAd5 / 35++ GFP and HDAd5 / 35++-SB / Flp at a 1:1 ratio at 6.25 x 1012GC / kg, and NHP #4 (Male), #5 (Female), and #6 (Female) were dosed with HDAd5 / 35++ GFP and HDAd5 / 35++-SB / Flp at a 1:1 ratio at 9.25 x 1012GC / kg. Arrow indicates time of HDAd administration. The PLT reference range (103 / µl) is 247-647 (Males) / 277-564 (Females). Data from one animal removed due to baseline abnormality.

[0127] Figure 39. Graphs showing complement analysis of Bb in (A), C2a in (B), and CH50 (C). NHP #1 (Female) was dosed with vehicle only, NHP #2 (Male) and #3 (Female) were dosed with HDAd5 / 35++ GFP and HDAd5 / 35++-SB / Flp at a 1:1 ratio at 6.25 x 1012GC / kg, and NHP #4 (Male), #5 (Female), and #6 (Female) were dosed with HDAd5 / 35++ GFP and HDAd5 / 35++-SB / Flp at a 1:1 ratio at 9.25 x 1012GC / kg. Arrow indicates time of HDAd administration. Arrow indicates time of HDAd administration. Data from one animal removed due to baseline abnormality.

[0128] Figure 40. Graphs showing serum cytokine analysis of interleukin-6 (IL-6) in (A) and monocyte chemoattractant protein-1 (MCP-1) in (B) for non-human primates (NHPs) dosed with either vehicle or HDAd5 / 35++ vector. NHP #1 (Female) was dosed with vehicle only, NHP #2 (Male) and #3 (Female) were dosed with HDAd5 / 35++ GFP and HDAd5 / 35++-SB / Flp at a 1:1 ratio at 6.25 x 1012GC / kg, and NHP #4 (Male), #5 (Female), and #6 (Female) were dosed with HDAd5 / 35++ GFP and HDAd5 / 35++- SB / Flp at a 1:1 ratio at 9.25 x 1012GC / kg. Arrow indicates time of HDAd administration. Arrow indicates time of HDAd administration. Data from one animal removed due to baseline abnormality.

[0129] Figure 41. Graph showing biodistribution analysis of HDAd5 / 35++ in NHPs dosed with HDAd5 / 35++ GFP and HDAd5 / 35++-SB / Flp at a 1:1 ratio at 9.25 x 1012GC / kg. Each triangle representsdata from one animal.

[0130] Figure 42. Graph showing biodistribution analysis of MGMT expression in NHPs dosed with HDAd5 / 35++ GFP and HDAd5 / 35++-SB / Flp at a 1:1 ratio at 9.25 x 1012GC / kg. Each triangle represents data from one animal. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS Definitions

[0131] As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001.

[0132] As used herein in the present disclosure, unless otherwise clear from context, (i) the term “a” or “an” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising”, “comprise”, “including” (whether used with “not limited to” or not), and “include” (whether used with “not limited to” or not) may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; (iv) the term “another” may be understood to mean at least an additional / second one or more; (v) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (vi) where ranges are provided, endpoints are included.

[0133] About: As used herein, the term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0134] Administering: As used herein, the term “administering” or “administration” typically refers to the administration of a composition to a subject to achieve delivery of an agent that is, or is included in, a composition to a target site or a site to be treated. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be parenteral. In some embodiments, administration may be intravenous. In some embodiments, administration may be oral. In some embodiments, administration may be via injection. In some embodiments, administration may besystemic. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., infusion, perfusion) for at least a selected period of time.

[0135] Agent: The term “agent” as used herein may refer to a compound or entity of any chemical class including, for example, polypeptides, nucleic acids, saccharides, lipids, small molecules, metals, or combinations thereof. As will be clear from context, in some embodiments, an agent can be or comprise a cell or organism, or a fraction, extract, or component thereof. In some embodiments, an agent is or comprises a natural product in that it is found in and / or is obtained from nature. In some embodiments, an agent is or comprises one or more entities that is man-made in that it is designed, engineered, and / or produced through action of the hand of man and / or is not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents are provided as collections or libraries, for example, that may be screened to identify or characterize active agents within them. Some particular embodiments of agents that may be utilized in accordance with the present disclosure include small molecules, antibodies, antibody fragments, aptamers, siRNAs, shRNAs, miRNAs, DNA / RNA hybrids, antisense oligonucleotides, ribozymes, peptides, peptide mimetics, small molecules, etc. In some embodiments, an agent is or comprises a polymer. In some embodiments, an agent is not a polymer and / or is substantially free of any polymer. In some embodiments, an agent contains at least one polymeric moiety. In some embodiments, an agent lacks or is substantially free of any polymeric moiety.

[0136] Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In some embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish and / or worms. In some embodiments, an animal may be a transgenic animal, a genetically- engineered animal and / or a clone.

[0137] Antibody: As used herein, the term "antibody" refers to an immunoglobulin molecule, or fragment thereof, that binds specifically to an epitope (e.g., of an antigen). Naturally-occurring human antibodies typically include two identical heavy chains and two identical light chains, each of which includes a variable domain and a constant domain. The constant domain defines the antibody isotype (IgG, IgA, IgM, IgE, etc.). Those skilled in the art are aware that different animal species utilize different antibody structures in nature. For example, camelid antibodies are single chain antibodies. Those skilled in the artwill further be aware that antibody variable domains are typically characterized by framework region (FR) sequences and complement-determining-region (CDR) sequences. Each variable domain typically includes three CDRs – CDR1, CDR2, and CDR3, which together contribute to specificity and / or affinity of epitope binding. In some embodiments, antibodies may be produced in or by organisms. In some embodiments, antibodies may be produced in or by cells in vitro (e.g., by hybridomas and / or by engineered cells). In some embodiments, antibody fragment(s) (e.g., as may be produced by cleavage or recombinantly) may be generated and / or utilized in accordance with the present disclosure. Those skilled in the art are aware that a variety of technologies have been developed to incorporate binding features (e.g., one or more CDRs and / or FR sequences, and in particular sets of 3 CDRs, optionally together with FR sequences) into new contexts. In some embodiments, CDR sequences may be maintained and other elements changed – e.g., as is done in humanization. Alternatively, or additionally, in some embodiments, variable regions may be associated with alternative constant regions (e.g., with constant regions from a different organism and / or that include one or more particular sequence features or elements desired, for example, to impart a particular attribute to the antibody agent. Those skilled in the art are further aware of a variety of technologies commonly utilized to associate binding features of two or more different antibodies with one another – e.g., in a single multispecific (most commonly bi-specific) agent. Various known formats of antibody agents (i.e., agents that incorporate antibody binding sequences such as one or more CDRs, or a full set of CDRs) include, for instance: antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); camelid antibodies; masked antibodies (e.g., Probodies); Small Modular ImmunoPharmaceuticals (“SMIPsTM”); single chain or Tandem diabodies (TandAb); VHHs; Anticalins; Nanobodies minibodies; BiTEs; ankyrin repeat proteins or DARPINs; Avimers; DARTs; TCR-like antibodies; Adnectins; Affilins; Trans-bodies; Affibodies; TrimerX; MicroProteins; Fynomers, Centyrins; KALBITORs, Zybodies, etc.

[0138] Binding: As used herein, the term “binding” typically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in one or more of a variety of contexts – for example, in some embodiments where interacting entities or moieties are studied in isolation, and / or in some embodiments where interacting entities or moieties are studied in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and / or in a biological system or cell). Binding between two entities may be considered “specific” if, under the conditions assessed, the relevant entities are more likely to associate with one another than with other available binding partners. Those skilled in the art will be aware of circumstances where a particular degreeof preference for one potential binding partner over another is required for binding to be deemed sufficiently “specific” for a particular purpose or situation.

[0139] CD46-binding adenoviral capsid: As used herein, the term “CD46-binding adenoviral capsid” refers to a capsid that includes a fiber or a portion thereof, e.g., a fiber knob that binds CD46. In some embodiments, a fiber or a portion thereof, e.g., a fiber knob that binds CD46 is derived from a Group B adenovirus, e.g., from Ad3, 7, 11, 14, 16, 21, 34, 35, 50, or 55. In some embodiments, a CD46-binding adenoviral capsid includes a fiber knob that binds CD46, e.g., a fiber knob derived from Ad3, 7, 11, 14, 16, 21, 34, 35, 50, or 55. In some embodiments, a CD46-binding adenoviral capsid includes a hexon and / or penton derived from Ad5, 6, or 35. In some embodiments, an adenoviral vector includes a chimeric Ad5 / 3, Ad5 / 7, Ad5 / 11, Ad5 / 14, Ad5 / 16, Ad5 / 21, Ad5 / 34, Ad5 / 35, Ad5 / 50, Ad5 / 55 or Ad6 / 35 capsid, e.g., with a hexon and / or penton derived from Ad5 or Ad6 and a fiber, fiber tail and / or fiber knob derived from Ad3, 7, 11, 14, 16, 21, 34, 35, 50, or 55.

[0140] Identity: As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., oligonucleotides, DNA, RNA, etc.) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCGsoftware package using an NWSgapdna.CMP matrix.

[0141] Immunosuppressive agent: As used herein, the term “immunosuppressive agent,” in its broadest sense, refers to an agent, e.g., a therapeutic agent that suppresses or reduces the activation, activity, or efficacy of the immune system of a subject. Exemplary immunosuppressive agents include, but are not limited to, abatacept, abrocitinib, adalimumab, alemtuzumab, anakinra, atacicept, azathioprine, baricitinib, basiliximab, belatacept, belimumab, bortezomib, certolizumab, crovalimab, cyclophosphamide, cyclosporine, daclizumab, dexamethasone, eculizumab, efalizumab, epratuzumab, etanercept, everolimus, fingolimod, fluorouracil, golimumab, hydroxychloroquine, imlifidase, infliximab, leflunomide, mercaptopurine, methotrexate, methylprednisolone, mycophenolate mofetil, mycophenolate sodium, ocrelizumab, ofatumumab, pimecrolimus, prednisone, prednisolone, ridaforolimus, rilonacept, rituximab, ruxolitinib, secukinumab, sirolimus, tacrolimus, temsirolimus, tocilizumab, tofacitinib, upadacitinib, and veltuzumab. Additional immunosuppressive agents are known in the art.

[0142] Immunosuppressive regimen: As used herein, the term “immunosuppressive regimen,” in its broadest sense, refers to a treatment regimen comprising one or more immunosuppressive agents.

[0143] In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within an organism (e.g., animal, plant and / or microbe).

[0144] In vivo: As used herein, the term “in vivo” refers to events that occur within an organism (e.g., animal, plant and / or microbe).

[0145] Nucleic acid: The term “nucleic acid”, as used herein, includes any nucleotides and polymers thereof. The term “polynucleotide”, as used herein, refers to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA) or a combination thereof. These terms refer to the primary structure of the molecules and, thus, include double- and single-stranded DNA, and double- and single-stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA comprising modified nucleotides and / or modified polynucleotides, such as, though not limited to, methylated, protected and / or capped nucleotides or polynucleotides. The terms encompass poly- or oligo-ribonucleotides (RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleobases and / or modified nucleobases; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified internucleotidic linkages. The term encompasses nucleic acids containing any combinations of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified internucleotidic linkages. Examples include, and are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties. Unless otherwise specified, the prefix poly- refers to a nucleic acid containing 2 to about 10,000nucleotide monomer units and wherein the prefix oligo- refers to a nucleic acid containing 2 to about 200 nucleotide monomer units.

[0146] One or more: As used herein, in some embodiments, “one or more” is 1-200, 1-150, 1-100, 1- 90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. In some embodiments, “one or more” is one. In some embodiments, “one or more” is two. In some embodiments, “one or more” is three. In some embodiments, “one or more” is four. In some embodiments, “one or more” is five. In some embodiments, “one or more” is six. In some embodiments, “one or more” is seven. In some embodiments, “one or more” is eight. In some embodiments, “one or more” is nine. In some embodiments, “one or more” is ten. In some embodiments, “one or more” is at least one. In some embodiments, “one or more” is at least two. In some embodiments, “one or more” is at least three. In some embodiments, “one or more” is at least four. In some embodiments, “one or more” is at least five. In some embodiments, “one or more” is at least six. In some embodiments, “one or more” is at least seven. In some embodiments, “one or more” is at least eight. In some embodiments, “one or more” is at least nine. In some embodiments, “one or more” is at least ten.

[0147] Patient: As used herein, the term “patient” refers to any organism to which a provided composition is or may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, a patient is a human. In some embodiments, a patient is suffering from or susceptible to one or more conditions, diseases, or disorders. In some embodiments, a patient displays one or more symptoms of a condition, disease, or disorder. In some embodiments, a patient has been diagnosed with one or more conditions, diseases, or disorders. In some embodiments, the patient is receiving or has received certain therapy to diagnose and / or to treat a condition, disease, or disorder.

[0148] Pharmaceutically acceptable: As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0149] Pharmaceutically acceptable carrier: As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptablecarriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and / or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0150] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, an active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0151] Polypeptide: The term “polypeptide”, as used herein, generally has its art-recognized meaning of a polymer of at least three amino acids. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional fragments (i.e., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term “polypeptide” as used herein. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a varietyof amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and / or characteristic portions thereof.

[0152] Promoter: As used herein, a “promoter” or “promoter sequence” can be a DNA regulatory region that directly or indirectly (e.g., through promoter-bound proteins or substances) participates in initiation and / or processivity of transcription of a coding sequence. A promoter may, under suitable conditions, initiate transcription of a coding sequence upon binding of one or more transcription factors and / or regulatory moieties with the promoter. A promoter that participates in initiation of transcription of a coding sequence can be “operably linked” to the coding sequence. In certain instances, a promoter can be or include a DNA regulatory region that extends from a transcription initiation site (at its 3’ terminus) to an upstream (5’ direction) position such that the sequence so designated includes one or both of a minimum number of bases or elements necessary to initiate a transcription event. A promoter may be, include, or be operably associated with or operably linked to, expression control sequences such as enhancer and repressor sequences. In some embodiments, a promoter may be inducible. In some embodiments, a promoter may be a constitutive promoter. In some embodiments, a conditional (e.g., inducible) promoter may be unidirectional or bi-directional. A promoter may be or include a sequence identical to a sequence known to occur in the genome of particular species. In some embodiments, a promoter can be or include a hybrid promoter, in which a sequence containing a transcriptional regulatory region can be obtained from one source and a sequence containing a transcription initiation region can be obtained from a second source. Systems for linking control elements to coding sequence within a transgene are well known in the art (general molecular biological and recombinant DNA techniques are described in Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0153] Subject: As used herein, the term “subject” or “test subject” refers to any organism to which a compound or composition is administered in accordance with the present disclosure e.g., for experimental, diagnostic, prophylactic and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, a subject is a human. In some embodiments, a subject may be suffering from and / or susceptible to a disease, disorder and / or condition.

[0154] Suffering from: An individual who is “suffering from” a disease, disorder, and / or condition has been diagnosed with and / or displays one or more symptoms of a disease, disorder, and / or condition.

[0155] Susceptible to: An individual who is “susceptible to” a disease, disorder and / or condition is one who has a higher risk of developing the disease, disorder and / or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition is predisposed to have that disease, disorder and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition may not have been diagnosed with the disease, disorder and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition may exhibit symptoms of the disease, disorder and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition may not exhibit symptoms of the disease, disorder and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0156] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen. In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of compound in a formulation to treat a disease, disorder, and / or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and / or reduces incidence of one or more symptoms or features of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0157] Treat: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and / or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0158] Vector: As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, whichrefers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into a viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” In some embodiments, the term “vector” refers to an agent capable of transporting a nucleic acid, wherein the agent comprises the nucleic acid. In some embodiments, a vector comprises or is an agent capable of transporting a nucleic acid.

[0159] Wild-type: As used herein, the term “wild-type” has its art-understood meaning that refers to an entity having a structure and / or activity as found in nature in a “normal” (as contrasted with mutant, diseased, altered, etc.) state or context. Those of ordinary skill in the art will appreciate that wild type genes and polypeptides often exist in multiple different forms (e.g., alleles). Description of Certain Embodiments

[0160] Among other things, the present disclosure provides vectors, genomes, and compositions thereof that may be used in methods of gene therapy, e.g., in vivo gene therapy. Particularly, the present disclosure provides adenoviral vectors, and genomes thereof, that can be used in methods of gene therapy, e.g., in vivo gene therapy.

[0161] Adenoviral vectors (and genomes thereof) are provided that may act as payload vectors for delivery of one or more therapeutic sequences to target cells. Various therapeutic sequences may be included in payload vectors of the present disclosure, including those disclosed herein. For example, therapeutic sequences encoding a therapeutic polypeptide are provided herein. In some embodiments, a therapeutic polypeptide comprises or is a CYBB, CYBA, NCF1, NCF2, NCF4, or CYBC1 therapeutic polypeptide. Payload vectors can also comprise a sequence encoding a selection marker that allows for the enrichment of modified target cells, e.g., target cells that have integrated a therapeutic sequence and a sequence encoding a selection marker. Further, payload vectors may include a variety of regulatory sequences that enable expression of a therapeutic sequence and / or a sequence encoding a selection marker in target cells. Payload vectors may include transposase target sites, and optionally, recombinase target sites that facilitate integration of a therapeutic sequence and / or a sequence encoding a selection marker.

[0162] Adenoviral vectors (and genomes thereof) are provided that may act as integration vectors for facilitation of integration of payload cassettes and / or selection cassettes from a payload vector into a target cell genome. Various integration-related polypeptides may be encoded by sequences included inintegration vectors of the present disclosure, including those disclosed herein. For example, an integration vector may comprise a sequence encoding a transposase, e.g., that can target transposase target sites in a payload vector. An integration vector may also comprise a sequence encoding a recombinase, e.g., that can target recombinase target sites in a payload vector. Further, integration vectors may include a variety of regulatory sequences that enable expression of a transposase and / or a recombinase in a target cell.

[0163] Payload vectors and integration vectors provided herein can be particularly useful for usage in methods of in vivo gene therapy. In vivo gene therapy can provide delivery of a therapeutic sequence to target cell populations in a subject. In some embodiments, a therapeutic sequence is integrated into target cell genomes in vivo. Methods of in vivo gene therapy provided herein can be useful for treatment of various conditions, disorders, and diseases. In some embodiments, a condition, disorder, or disease is an immune deficiency disorder. In some embodiments, a condition, disorder, or disease is chronic granulomatous disease (CGD).

[0164] The present disclosure includes compositions, e.g., pharmaceutical compositions that comprise one or more adenoviral vectors, e.g., a payload vector and / or an integration vector. Pharmaceutical compositions provided herein may contain a payload vector and an integration vector in a ratio of about 1- 3:1 to about 1:1-3 payload vector:integration vector. In some embodiments, pharmaceutical compositions are administered to a subject in a method, e.g., a method of in vivo gene therapy.

[0165] Adenoviral vectors of the present disclosure can be administered to a subject at various doses. The present disclosure recognizes that the determination of a dose based on genome copies, e.g., genome copies per kilogram (GC / kg) of a subject, can provide therapeutic effects. For example, in some embodiments, a dose is about 2.5 x 1011to about 1.25 x 1013GC / kg. In some embodiments, a dose of a payload vector and an integration vector is a combined dose of about 2.5 x 1011to about 1.25 x 1013GC / kg. A payload vector and an integration vector, e.g., in form of a pharmaceutical composition, can be administered to a subject as a single dose, e.g., of about 2.5 x 1011to about 1.25 x 1013GC / kg, on a single day and can provide therapeutic effects. For example, as detailed herein, a single dose of a pharmaceutical composition comprising a payload vector and an integration vector at a ratio of about 1-3:1 to about 1:1-3 payload vector:integration vector and a dose of about 2.5 x 1011to about 1.25 x 1013GC / kg can provide therapeutic effects in a subject, e.g., a mammalian subject.

[0166] One such condition, disorder, or disease that can be treated using adenoviral vectors and compositions thereof provided herein is a primary immunodeficiency disease, e.g., chronic granulomatous disease (CGD). CGD is reportedly an inherited immune deficiency disorder that results from mutations in one or more components of NADPH oxidase complex. In some embodiments, CGD is associated with one or more mutations in CYBB, CYBA, NCF1, NCF2, NCF4, and / or CYBC1. Disruption of NADPH oxidase complex activity reportedly leads to a lack of production of reactive oxygen species in certain immune cells,e.g., phagocytes, e.g., neutrophils. Accordingly, killing of bacteria and fungi by immune cells is impaired, leading to an increased susceptibility of a subject suffering from CGD to various bacterial and fungal infections.

[0167] Without being bound by any one theory, providing a therapeutic sequence encoding a wild- type CYBB, CYBA, NCF1, NCF2, NCF4, and / or CYBC1 polypeptide can be beneficial to a subject comprising one or more mutations in CYBB, CYBA, NCF1, NCF2, NCF4, and / or CYBC1. For example, a method of in vivo gene therapy provided herein can provide a therapeutic sequence encoding a CYBB, CYBA, NCF1, NCF2, NCF4, and / or CYBC1 therapeutic polypeptide to target cells, e.g., HSCs and cells derived therefrom, in a subject. In one embodiment, the in vivo therapy encodes one, two, three, four or all of the sequences encoding CYBB, CYBA, NCF1, NCF2, NCF4, and / or CYBC1. Expression of a CYBB, CYBA, NCF1, NCF2, NCF4, and / or CYBC1 therapeutic polypeptide in target cells of a subject can provide therapeutic effects including, e.g., increased NADPH oxidase activity. In some embodiments, the present disclosure provides a method of treatment of chronic granulomatous disease using technologies (e.g., vectors, genomes, compositions) provided herein. Adenoviral Vectors

[0168] Among other things, the present disclosure provides adenoviral vectors and genomes thereof that can be used in methods of gene therapy, e.g., in vivo gene therapy. Adenoviruses are large, icosahedral- shaped, non-enveloped viruses. Natural adenoviral capsids include three types of proteins: fiber, penton, and hexon. The hexon makes up the majority of the viral capsid, forming 20 triangular faces. A penton base is located at each of the 12 vertices of the capsid, and a fiber (also referred to as knobbed fiber) protrudes from each penton base. Penton and fiber, and in particular the fiber knob, are of particular importance in receptor binding and internalization as they facilitate the attachment of the capsid to host cells.

[0169] Adenoviral genomes include adenoviral DNA flanked on both ends by serotype-specific inverted terminal repeats (ITRs), which are understood to be cis elements that contribute to or are necessary for viral genome replication and packaging. Depending on the serotype, ITRs can be approximately 100- 200 base pairs (e.g., about 160 base pairs) in length, with highest conservation at nucleotide positions (e.g., ~50 base pairs) closest to the adenoviral genome termini. Adenoviral genomes also include a packaging sequence (e.g., a conditional or non-conditional packaging sequence), which can facilitate packaging of the viral genome into viral vectors. Packaging sequences are located in the left portion of the genome.

[0170] Natural adenoviral genomes encode several proteins including early transcriptional units, E1, E2, E3, and E4 and late transcriptional units which encode structural protein components of the adenoviral vector. Early (E) and late (L) transcription are divided by the onset of viral genome replication. The E1region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral genome replication. These proteins are involved in DNA replication, late gene expression, and host cell shut-off. The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP is particularly efficient during the late phase of infection. mRNAs transcribed using this promoter can include a 5'-tripartite leader (TPL) sequence that facilitates translation.

[0171] The present disclosure includes Ad5, 6, or 35 genomes. In various embodiments, an Ad5, 6, or 35 genome is a single-stranded or double-stranded DNA sequence that includes ITRs derived from the genome of an Ad5, 6, or 35 adenovirus (e.g., a 5′ ITR according to SEQ ID NO: 3, 23, or 42 and a 3′ ITR according to SEQ ID NO: 4, 24, or 43), or ITRs that individually and / or together have at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) thereto. In various embodiments, an Ad5, 6, or 35 genome is a single-stranded or double-stranded DNA sequence that includes a packaging sequence derived from the genome of an Ad5, 6, or 35 adenovirus (e.g., a packaging sequence according to SEQ ID NO: 5, 25, or 44), or a packaging sequence having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the entirety or a portion thereof. In various embodiments, an Ad5, 6, or 35 genome is a single-stranded or double- stranded DNA sequence that includes a sequence with at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to all, a portion of, or a contiguous corresponding portion of, or a discontiguous corresponding portion of a reference Ad5, 6, or 35 genome (e.g., SEQ ID NO: 1, 21, or 41).

[0172] In various embodiments, an Ad5, 6, or 35 genome is any nucleotide sequence that includes at least ITRs derived from the genome of an Ad5, 6, or 35 adenovirus (e.g., a 5′ ITR according to SEQ ID NO: 3, 23, or 42 and a 3′ ITR according to SEQ ID NO: 4, 24, or 43), or ITRs that individually and / or together have at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) thereto. In various embodiments, an Ad5, 6, or 35 genome is an Ad5, 6, or 35 genome from which one or more nucleotides, coding sequences, and / or genes are completely or partially deleted as compared to a reference sequence. For example, in some embodiments, an Ad5, 6, or 35 genome can be a genome that does not include one or more of E1, E2, E3, and E4. In certain embodiments, an Ad5, 6, or 35 genome is a genome that does not include any coding sequences of an Ad5, 6, or 35 genome (e.g., a “gutless” vector that includes ITRs having at least 75% sequence identity to Ad5, 6, or 35 genome ITRs but includes none of the coding sequences present in a reference Ad5, 6, or 35 genome).

[0173] In various embodiments, an Ad5, 6, or 35 genome includes, does not include, or includes a deletion of, all or a portion of an E1 sequence according to SEQ ID NO: 6, 26, 45, or a sequence having atleast 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) thereto.

[0174] In various embodiments, an Ad5, 6, or 35 genome includes, does not include, or includes a deletion of, all or a portion of an E2 sequence according to SEQ ID NO: 7, 27, 46, or a sequence having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) thereto.

[0175] In various embodiments, an Ad5, 6, or 35 genome includes, does not include, or includes a deletion of, all or a portion of an E3 sequence according to SEQ ID NO: 8, 28, 47, or a sequence having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) thereto.

[0176] In various embodiments, an Ad5, 6, or 35 genome includes, or does not include, a sequence that encodes a fiber, where the sequence has at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ ID NO: 9, 29, or 48.

[0177] In various embodiments, an Ad5, 6, or 35 genome includes, or does not include, a sequence that encodes a fiber tail, where the sequence has at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ ID NO: 10, 30, or 49.

[0178] In various embodiments, an Ad5, 6, or 35 genome includes, or does not include, a sequence that encodes a fiber shaft, where the sequence has at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ ID NO: 11, 31, or 50.

[0179] In various embodiments, an Ad5, 6, or 35 genome includes, or does not include, a sequence that encodes a fiber knob, where the sequence has at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ ID NO: 12, 32, or 51.

[0180] In various embodiments, an Ad5, 6, or 35 genome includes, or does not include, a sequence that encodes a penton, where the sequence has at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ ID NO: 13, 33, or 52.

[0181] In various embodiments, an Ad5, 6, or 35 genome includes, or does not include, a sequence that encodes a hexon, where the sequence has at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ ID NO: 14, 34, or 53.

[0182] The present disclosure includes adenoviral vectors that include a CD46-binding adenoviral capsid. In some embodiments, a CD46-binding adenoviral capsid includes a fiber or a portion thereof, e.g., a fiber knob that binds CD46. In some embodiments, a fiber or a portion thereof, e.g., a fiber knob that binds CD46 is derived from a Group B adenovirus, e.g., from Ad3, 7, 11, 14, 16, 21, 34, 35, 50, or 55. In some embodiments, a CD46-binding adenoviral capsid includes a fiber knob that binds CD46, e.g., a fiber knob derived from Ad3, 7, 11, 14, 16, 21, 34, 35, 50, or 55. In some embodiments, a CD46-bindingadenoviral capsid includes a hexon and / or penton derived from Ad5, 6, or 35. In some embodiments, an adenoviral vector includes a chimeric Ad5 / 3, Ad5 / 7, Ad5 / 11, Ad5 / 14, Ad5 / 16, Ad5 / 21, Ad5 / 34, Ad5 / 35, Ad5 / 50, Ad5 / 55 or Ad6 / 35 capsid, e.g., with a hexon and / or penton derived from Ad5 or Ad6 and a fiber, fiber tail and / or fiber knob derived from Ad3, 7, 11, 14, 16, 21, 34, 35, 50, or 55.

[0183] In some embodiments, a fiber knob may include mutations that increase the affinity to CD46. For example, in some embodiments, an Ad35 fiber knob of an Ad35 vector or chimeric Ad vector that includes an Ad35 fiber knob is a mutant Ad35 fiber knob. In particular embodiments, a mutant Ad35 fiber knob is an Ad35++ mutant fiber knob (alternatively referred to herein as an Ad35++ fiber knob). In various embodiments, an Ad35++ mutant fiber knob is an Ad35 fiber knob mutated to increase the affinity to CD46, e.g., by 25-fold, e.g., such that the Ad35++ mutant fiber knob increases cell transduction efficiency, e.g., at lower multiplicity of infection (MOI) (Li and Lieber, FEBS Letters, 593(24): 3623-3648, 2019). In various embodiments, an Ad35++ mutant fiber knob includes at least one mutation selected from Ile192Val, Asp207Gly (or Glu207Gly in certain Ad35 sequences), Asn217Asp, Thr226Ala, Thr245Ala, Thr254Pro, Ile256Leu, Ile256Val, Arg259Cys, and Arg279His. In various embodiments, an Ad35++ mutant fiber knob includes each of the following mutations: Ile192Val, Asp207Gly (or Glu207Gly in certain Ad35 sequences), Asn217Asp, Thr226Ala, Thr245Ala, Thr254Pro, Ile256Leu, Ile256Val, Arg259Cys, and Arg279His. In various embodiments, amino acid numbering of an Ad35 fiber is according to GenBank Accession No. AP_000601 (SEQ ID NO: 54) or an amino acid sequence corresponding thereto, e.g., where position 207 is Glu or Asp. In various embodiments, an Ad35 fiber has an amino acid sequence according to GenBank Accession No. AP_000601 (SEQ ID NO: 54). Further description of Ad35++ fiber knob mutations is found in Wang 2008 J. Virol.82(21): 10567–10579, which is incorporated herein by reference in its entirety and with respect to fiber knobs. The present disclosure includes, for example, a recombinant Ad35 vector with a mutant Ad35 fiber knob, an Ad5 / 35 vector with a mutant Ad35 fiber knob, or an Ad6 / 35 vector with a mutant Ad35 fiber knob.

[0184] The present disclosure includes Ad5 / 3, Ad5 / 7, Ad5 / 11, Ad5 / 14, Ad5 / 16, Ad5 / 21, Ad5 / 34, Ad5 / 35, Ad5 / 50, Ad5 / 55, Ad6 / 35 or Ad35 vectors that include a fiber having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an Ad3, 7, 11, 14, 16, 21, 34, 35, 50, or 55 fiber (e.g., a fiber according to SEQ ID NO: 60, 62, 64, 66, 68, 70, 72, 54, 74, or 76).

[0185] The present disclosure includes Ad5 / 3, Ad5 / 7, Ad5 / 11, Ad5 / 14, Ad5 / 16, Ad5 / 21, Ad5 / 34, Ad5 / 35, Ad5 / 50, Ad5 / 55, Ad6 / 35 or Ad35 vectors that include a fiber tail having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an Ad3, 7, 11, 14, 16, 21, 34, 35, 50, or 55 fiber tail (where the fiber tail is the portion of the fiber including all amino acids N-terminal to the fiber shaft).

[0186] The present disclosure includes Ad5 / 3, Ad5 / 7, Ad5 / 11, Ad5 / 14, Ad5 / 16, Ad5 / 21, Ad5 / 34, Ad5 / 35, Ad5 / 50, Ad5 / 55, Ad6 / 35 or Ad35 vectors that include a fiber shaft having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an Ad3, 7, 11, 14, 16, 21, 34, 35, 50, or 55 fiber shaft.

[0187] The present disclosure includes Ad5 / 3, Ad5 / 7, Ad5 / 11, Ad5 / 14, Ad5 / 16, Ad5 / 21, Ad5 / 34, Ad5 / 35, Ad5 / 50, Ad5 / 55, Ad6 / 35 or Ad35 vectors that include a fiber knob having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an Ad3, 7, 11, 14, 16, 21, 34, 35, 50, or 55 fiber knob (e.g., a fiber knob according to SEQ ID NO: 61, 63, 65, 67, 69, 71, 73, 57, 75, or 77).

[0188] The present disclosure includes Ad5 / 3, Ad5 / 7, Ad5 / 11, Ad5 / 14, Ad5 / 16, Ad5 / 21, Ad5 / 34, Ad5 / 35, Ad5 / 50, Ad5 / 55, Ad6 / 35 or Ad35 vectors that include a penton having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an Ad5, 6, or 35 penton (e.g., a penton according to SEQ ID NO: 19, 39, or 58).

[0189] The present disclosure includes Ad5 / 3, Ad5 / 7, Ad5 / 11, Ad5 / 14, Ad5 / 16, Ad5 / 21, Ad5 / 34, Ad5 / 35, Ad5 / 50, Ad5 / 55, Ad6 / 35 or Ad35 vectors that include a hexon having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an Ad5, 6, or 35 hexon (e.g., a hexon according to SEQ ID NO: 20, 40, or 59). In some embodiments, an Ad5, 6, or 35 hexon may be chimeric, i.e., it may include hexon sequences, e.g., HVR sequences including one or more of the HVR1-7 sequences, from a different adenoviral serotype, e.g., from a simian adenovirus.

[0190] In some embodiments, an adenoviral vector of the present disclosure, e.g., an Ad5 / 3, Ad5 / 7, Ad5 / 11, Ad5 / 14, Ad5 / 16, Ad5 / 21, Ad5 / 34, Ad5 / 35, Ad5 / 50, Ad5 / 55, Ad6 / 35 or Ad35 vector, can be a chimeric adenoviral vector that includes at least a fiber knob having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an Ad3, 7, 11, 14, 16, 21, 34, 35, 50, or 55 fiber knob and at least one protein or portion thereof (such as a fiber shaft, fiber tail, penton, or hexon) that has at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the protein or portion thereof (such as a fiber shaft, fiber tail, penton, or hexon) of a different adenoviral serotype.

[0191] In some embodiments, an adenoviral vector of the present disclosure, e.g., an Ad5 / 3, Ad5 / 7, Ad5 / 11, Ad5 / 14, Ad5 / 16, Ad5 / 21, Ad5 / 34, Ad5 / 35, Ad5 / 50, Ad5 / 55, Ad6 / 35 or Ad35 vector, can be a chimeric adenoviral vector that includes at least a fiber shaft having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an Ad3, 7, 11, 14, 16, 21, 34, 35, 50, or 55 fiber shaft and at least one protein or portion thereof (such as a fiber knob, fiber tail, penton, or hexon) that has at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% identity) to the protein or portion thereof (such as a fiber knob, fiber tail, penton, or hexon) of a different adenoviral serotype.

[0192] In some embodiments, an adenoviral vector of the present disclosure, e.g., an Ad5 / 3, Ad5 / 7, Ad5 / 11, Ad5 / 14, Ad5 / 16, Ad5 / 21, Ad5 / 34, Ad5 / 35, Ad5 / 50, Ad5 / 55, Ad6 / 35 or Ad35 vector, can be a chimeric adenoviral vector that includes at least a fiber tail having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an Ad3, 7, 11, 14, 16, 21, 34, 35, 50, or 55 fiber tail and at least one protein or portion thereof (such as a fiber knob, fiber shaft, penton, or hexon) that has at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the protein or portion thereof (such as a fiber knob, fiber shaft, penton, or hexon) of a different adenoviral serotype.

[0193] In some embodiments, an adenoviral vector of the present disclosure, e.g., an Ad5 / 3, Ad5 / 7, Ad5 / 11, Ad5 / 14, Ad5 / 16, Ad5 / 21, Ad5 / 34, Ad5 / 35, Ad5 / 50, Ad5 / 55, Ad6 / 35 or Ad35 vector, can be a chimeric adenoviral vector that includes at least a penton having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an Ad5, 6, or 35 penton and at least one protein or portion thereof (such as a fiber knob, fiber shaft, fiber tail, or hexon) that has at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the protein or portion thereof (such as a fiber knob, fiber shaft, fiber tail, or hexon) of a different adenoviral serotype.

[0194] In some embodiments, an adenoviral vector of the present disclosure, e.g., an Ad5 / 3, Ad5 / 7, Ad5 / 11, Ad5 / 14, Ad5 / 16, Ad5 / 21, Ad5 / 34, Ad5 / 35, Ad5 / 50, Ad5 / 55, Ad6 / 35 or Ad35 vector, can be a chimeric adenoviral vector that includes at least a hexon having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an Ad5, 6, or 35 hexon and at least one protein or portion thereof (such as a fiber knob, fiber shaft, fiber tail, or penton) that has at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the protein or portion thereof (such as a fiber knob, fiber shaft, fiber tail, or penton) of a different adenoviral serotype.

[0195] Exemplary sequences of Ad5, 6, or 35 components (e.g., ITRs, packaging sequences, genes, and proteins) are provided in the following tables. Viral polypeptides include proteins that are components of viral vectors and portions or fragments thereof, including for example a fiber, fiber knob, fiber shaft, fiber tail, penton, or hexon. Exemplary Ad3, 7, 11, 14, 16, 21, 34, 35, 50, or 55 fiber sequences (including fiber knob sequences) are also provided in the following tables.

[0196] In some embodiments, a vector, e.g., an adenoviral vector, e.g., a HDAd vector, of the present disclosure includes Ad5 capsid proteins except that the fibers are chimeric in that they include an Ad5 fiber tail, an Ad5 or Ad35 fiber shaft, and an Ad35 fiber knob (see, e.g., Shayakhmetov et al. 2000 J. Virol74(6):2567-2583, which is hereby incorporated by reference in its entirety), optionally where the Ad35 fiber knob is mutated for increased affinity to CD46 (e.g., Ad5 / 35++). In some embodiments, an Ad5 / 35++ vector is a chimeric Ad5 / 35 vector with a mutant Ad35++ fiber knob (see, e.g., Wang et al.2008 J. Virol. 82(21):10567-79, which is incorporated herein by reference in its entirety). In some embodiments, an Ad35++ mutant fiber knob is an Ad35 fiber knob mutated to increase the affinity to CD46, e.g., by 25-fold, e.g., such that the Ad35++ mutant fiber knob increases cell transduction efficiency, e.g., at lower multiplicity of infection (MOI) (Li and Lieber, FEBS Letters, 593(24): 3623-3648, 2019, which is hereby incorporated by reference). It is to be understood that reference to an Ad5 / 35++ vector herein, e.g., an HDAd5 / 35++ vector may only include an Ad35++ fiber knob with all other proteins from Ad5 (optionally with certain modifications, e.g., in the case of the Ad5 hexon protein it may include one or more HVR swaps with one or more HVRs from another adenoviral hexon, e.g., from a simian adenovirus). In some embodiments, an HDAd5 / 35++ vector may further include an Ad35 shaft.

[0197] In some embodiments, a vector, e.g., an adenoviral vector, e.g., a HDAd vector, of the present disclosure includes Ad6 capsid proteins except that the fibers are chimeric in that they include an Ad6 fiber tail, an Ad6 or Ad35 fiber shaft, and an Ad35 fiber knob (see, e.g., Wang et al.2023 Mol. Ther.29:P213- 226, which is hereby incorporated by reference in its entirety), optionally where the Ad35 fiber knob is mutated for increased affinity to CD46 (e.g., Ad6 / 35++). In some embodiments, an Ad6 / 35++ vector is a chimeric Ad6 / 35 vector with a mutant Ad35++ fiber knob (see, e.g., Wang et al. 2008 J. Virol. 82(21):10567-79, which is incorporated herein by reference in its entirety). In some embodiments, an Ad35++ mutant fiber knob is an Ad35 fiber knob mutated to increase the affinity to CD46, e.g., by 25-fold, e.g., such that the Ad35++ mutant fiber knob increases cell transduction efficiency, e.g., at lower multiplicity of infection (MOI) (Li and Lieber, FEBS Letters, 593(24): 3623-3648, 2019, which is hereby incorporated by reference). It is to be understood that reference to an Ad6 / 35++ vector herein, e.g., a HDAd6 / 35++ vector may only include an Ad35++ fiber knob with all other proteins from Ad6 (optionally with certain modifications). In some embodiments, an HDAd6 / 35++ vector may further include an Ad35 shaft.

[0198] In some embodiments, an adenoviral vector or genome of the present disclosure can be an adenoviral vector and / or genome disclosed in WO 2021 / 003432, which is herein incorporated by reference in its entirety, and particularly with respect to adenoviral vectors and genomes.

[0199] Various sequences corresponding to accession numbers disclosed herein, including e.g., accession numbers referred to herein as SEQ ID NOs: 1, 21, and 41 as indicated in Tables 1, 3 and 5 are provided herein in the below listing of accession sequences. Those of skill in the art will appreciate that such sequences, including the sequences disclosed in the below listing of accession sequences, can bereferenced in whole (e.g., by an accession number) or in part (e.g., by reference to a nucleotide position and / or a set or range of nucleotide positions of a sequence and / or accession number). Table 1: Ad5 Genomic SequencesTable 2: Ad5 Amino Acid SequencesTable 3: Ad6 Genomic SequencesTable 4: Ad6 Amino Acid SequencesTable 5: Ad35 Genomic SequencesTable 6: Ad35 Amino Acid SequencesTable 7: Other Group B Fiber Sequences Fiber Amino Acid SequencesManufacturing

[0200] Among other things, the present disclosure provides various methods for producing adenoviral vectors and genomes provided herein.

[0201] In various embodiments, a vector of the present disclosure is a helper-dependent adenoviral vector (HDAd or “third generation” vector). Third generation adenoviral vectors can also be referred to as gutless. HDAd vectors are engineered to remove all viral coding sequences from the reference Ad sequence and retain only the ITRs of the genome and the packaging sequence of the genome or a functional fragment thereof. Retained portions of the reference genome can be identical in sequence to a reference genome or can have less than 100% identity with a reference genome, e.g., at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% identity.

[0202] Because HDAd vector genomes do not encode the proteins necessary for viral production, they are “helper-dependent.” A helper-dependent genome can only be packaged into a vector if present in a cell that includes nucleic acid sequences that provides the necessary viral proteins in trans. These helper- dependent vectors are characterized by greater capacity than first- and second-generation adenoviral vectors that retain sequences encoding certain viral proteins. Because HDAd vectors do not express viral geneswhen used as a vector, they are also less immunogenic and the risk of cytotoxicity or interferon response in recipients is reduced.

[0203] It has been observed that the certain HDAd vector genomes can be most efficiently packaged when the genome has at least a minimum a total length, e.g., a minimum to total length of at least 20 kb (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 kb) which length can include, e.g., a “payload” and / or a “stuffer” sequence. Where a payload does not utilize a number of nucleotides that causes the adenoviral genome to have at least a target length, the stuffer sequence can be used to achieve or surpass the target length. A minimum length for efficient packaging is not required for beneficial use of vectors provided herein, such that meeting any target length may be advantageous but not required for use of compositions and methods provided herein. Like other adenoviral vectors, typical HDAd genomes generally remain episomal and do not integrate with a host genome.

[0204] As noted previously, because HDAd vectors do not encode the viral proteins required to produce viral particles, viral proteins must be provided in trans, e.g., expressed in and / or by cells in which the HDAd genome is present. In some HDAd vector systems, one viral genome (a helper genome) encodes all of the proteins for replication (e.g., all of the structural viral proteins) required but has a conditional defect in the packaging sequence, making it less likely to be packaged into an adenoviral vector under certain vector production conditions (e.g., in the presence of an agent that reduces function of the conditionally defective packaging sequence). Thus, the HDAd genome includes (e.g., only includes) 5’ and 3’ ITRs, a payload, and a functional packaging sequence (e.g., a wild-type packaging sequence or a functional fragment thereof), which allows the HDAd genome to be selectively packaged into HDAd vectors produced from structural viral proteins expressed from the helper genome. Production of HDAd vectors can include co-transfection of a plasmid containing the HDAd genome and a packaging-defective helper virus that provides viral proteins. The helper virus can rescue propagation of the HDAd genome and HDAd vectors can be produced, e.g., at a large scale, and isolated. Various protocols are known in the art, e.g., Palmer et al., Gene Therapy Protocols: Methods in Molecular Biology 2009;433:33.

[0205] In various embodiments, a helper genome can include deletion of E1, e.g., where the helper genome includes all of the viral genes except for E1 and E1 expression products are supplied by complementary expression from the genome of the producer cell line. In some embodiments, to prevent generation of replication competent adenoviral vectors (RCA) as a consequence of homologous recombination between the helper and HDAd genomes present in producer cells, a “stuffer” sequence can be inserted into the E3 region of the helper genome to render any recombinants too large to be packaged and / or efficiently packaged.

[0206] In some HDAd vector systems, a helper genome utilizes a recombinase system (e.g., a Cre / loxP system) for conditional packaging. In certain such HDAd vector systems, a helper genome can include apackaging sequence or functional fragment thereof (e.g., a fragment of the packaging sequence that is sufficient for packaging, required for packaging, or required for efficient packaging of the Ad genome into the capsid) flanked by recombinase (e.g., loxP) sites so that contact with a corresponding recombinase (e.g., Cre recombinase) excises the packaging sequence or functional fragment thereof from the helper genome by recombinase-mediated (e.g., Cre-mediated) site-specific recombination between the recombinase target sites (e.g., loxP sites). For production of HDAd vectors, an HDAd genome can be delivered to cells that express a recombinase for excision of the conditional packaging sequence of a helper vector (e.g., HEK293 cells that expresses Cre recombinase), optionally where the HDAd genome is delivered to the cells in a non-viral vector form, such as a bacterial plasmid form (e.g., where the HDAd donor genome is present in a bacterial plasmid and / or is liberated by restriction enzyme digestion). The same cells can be transduced with the helper genome including a packaging sequence or functional fragment thereof flanked by recombinase target sites (e.g., loxP sites). Thus, producer cells can be transfected with the HDAd genome and transduced with a helper genome bearing a packaging sequence or a functional fragment thereof flanked by recombinase target sites (e.g., loxP sites), where the cells express a recombinase (e.g., Cre) corresponding to the recombinase target sites such that excision of the packaging sequence or functional fragment thereof renders the helper virus genome deficient for packaging (e.g., unpackageable), but still able to provide all of the necessary trans-acting factors for production of HDAd vector including the HDAd genome.

[0207] Similar HDAd production systems have been developed using Flp / Frt (e.g., FlpE / Frt, e.g., Flpx9 / Frt) site-specific recombination, where Flp-mediated recombination between Frt sites flanking the packaging sequence or functional fragment thereof of the helper genome reduces or eliminates packaging of helper genomes in producer cells that express Flp.

[0208] HDAd vectors including the HDAd genome including the payload can be isolated from the producer cells. HDAd vectors can be further purified from helper vectors by physical means. In general, some contamination of helper vectors and / or helper genomes in HDAd vectors and HDAd vector formulations can occur and can be tolerated. In some embodiments, a preparation of HDAd vectors, e.g., HDAd payload vectors and / or HDAd integration vectors, comprises about 3% or less helper vectors as based on, e.g., genome copies determined by dPCR. In some embodiments, a preparation of HDAd vectors, e.g., HDAd payload vectors and / or HDAd integration vectors, comprises about 2% or less helper vectors as based on, e.g., genome copies determined by dPCR. In some embodiments, a preparation of HDAd vectors, e.g., HDAd payload vectors and / or HDAd integration vectors, comprises about 1% or less helper vectors as based on, e.g., genome copies determined by dPCR. In some embodiments, a preparation of HDAd vectors, e.g., HDAd payload vectors and / or HDAd integration vectors, comprises about 0.5% or less helper vectors as based on, e.g., genome copies determined by dPCR. In some embodiments, a preparationof HDAd vectors, e.g., HDAd payload vectors and / or HDAd integration vectors, comprises about 0.1% or less helper vectors as based on, e.g., genome copies determined by dPCR.

[0209] An additional optional engineering consideration can be engineering of a helper genome to have a size that permits separation of helper vector from HDAd vector by centrifugation, e.g., by CsCl ultracentrifugation. One means of achieving this result is to increase (or decrease) the size of the helper genome as compared to the HDAd genome. Payload Vectors

[0210] Among other things, the present disclosure provides payload vectors that comprise an adenoviral capsid that targets CD46+ cells, e.g., an Ad5 / 35++ adenoviral capsid or any of the other CD46 targeting capsids described herein and a double stranded DNA genome comprising (i) a 5’ ITR and a 3’ ITR, (ii) a packaging sequence, (iii) a payload cassette comprising a therapeutic sequence and a promoter operably linked to the therapeutic sequence, (iv) a selection cassette comprising a nucleic acid sequence encoding a selection marker and a promoter operably linked to the nucleic acid sequence encoding a selection marker, (v) a first transposase target site and a second transposase target site, and, optionally, (iv) a first recombinase target site and a second recombinase target site. In some embodiments, the payload vector is a HDAd payload vector.

[0211] The present disclosure provides payload vectors, e.g., adenoviral payload vectors, e.g., HDAd payload vectors, that comprise one or more payload cassettes comprising one or more therapeutic sequences. Payload vectors, e.g., adenoviral payload vectors, e.g., HDAd payload vectors, generally are used for delivery of a payload, e.g., one or more therapeutic sequences, to target cells in vitro, ex vivo, or in vivo. In some embodiments, a HDAd payload vector is used in a method, e.g., a method of in vivo gene therapy, provided herein. In some embodiments, a HDAd payload vector is used in combination with a HDAd integration vector provided herein.

[0212] In some embodiments, a payload vector comprises one or more payload cassettes, e.g., one or more payload cassettes provided herein, and a selection cassette, e.g., a selection cassette provided herein. In some embodiments, a payload vector comprises a genome comprising one or more payload cassettes, e.g., one or more payload cassettes provided herein, and a selection cassette, e.g., a selection cassette provided herein. In some embodiments, a payload vector comprises a payload cassette and a selection cassette. In some embodiments, a payload vector comprises a genome that comprises a payload cassette and a selection cassette. 5’ ITR and 3’ ITR

[0213] Adenoviral genomes consist of linear DNA molecules containing inverted terminal repeatsequences (ITRs) of 100 to 200 base pairs. Adenoviral ITRs contain a number of cis-acting elements that are involved in the initiation of viral DNA replication. In some embodiments, a payload vector comprises a 5’ ITR and a 3’ ITR that are of the same serotype as the adenoviral capsid.

[0214] In some embodiments, the 5’ ITR and the 3’ ITR are Ad5 ITRs and the adenoviral capsid is an Ad5 / 35 or Ad5 / 35++ adenoviral capsid or an Ad5 / 3, Ad5 / 7, Ad5 / 11, Ad5 / 14, Ad5 / 16, Ad5 / 21, Ad5 / 34, Ad5 / 50 or Ad5 / 55 adenoviral capsid. In some embodiments, a payload vector comprises an Ad55’ ITR and an Ad53’ ITR. In some embodiments, an Ad55’ ITR comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 3. In some embodiments, an Ad55’ ITR comprises a sequence of SEQ ID NO: 3. In some embodiments, an Ad53’ ITR comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 4. In some embodiments, an Ad53’ ITR comprises a sequence of SEQ ID NO: 4.

[0215] In some embodiments, the 5’ ITR and the 3’ ITR are Ad6 ITRs and the adenoviral capsid is an Ad6 / 35 or Ad6 / 35++ adenoviral capsid. In some embodiments, a payload vector comprises an Ad65’ ITR and an Ad63’ ITR. In some embodiments, an Ad65’ ITR comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 23. In some embodiments, an Ad65’ ITR comprises a sequence of SEQ ID NO: 23. In some embodiments, an Ad63’ ITR comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 24. In some embodiments, an Ad63’ ITR comprises a sequence of SEQ ID NO: 24.

[0216] In some embodiments, the 5’ ITR and the 3’ ITR are Ad35 ITRs and the adenoviral capsid is an Ad35 or Ad35++ adenoviral capsid. In some embodiments, a payload vector comprises an Ad355’ ITR and an Ad353’ ITR. In some embodiments, an Ad355’ ITR comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 42. In some embodiments, an Ad35 5’ ITR comprises a sequence of SEQ ID NO: 42. In some embodiments, an Ad353’ ITR comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 43. In some embodiments, an Ad35 3’ ITR comprises a sequence of SEQ ID NO: 43.

[0217] Those skilled in the art will be able to select suitable ITRs from the genomes of Ad5 and other adenoviral serotypes, e.g., from the genomes of Ad6 or Ad35, e.g., by reference to GenBank Accession No. AC_000008.1 (Ad5), GenBank Accession No. OP871032.1 (Ad6), and GenBank Accession No. AC_000019.1 (Ad35). Table 8: Exemplary ITR SequencesPackaging sequence

[0218] Adenoviral genomes carry a unique sequence, called the packaging sequence that comprises a series of AT-rich repeats and is normally located between the 5’ ITR and the E1A transcription start site. The packaging sequence is recognized by adenoviral proteins involved in packaging the adenoviral genome into the adenoviral capsid. In some embodiments, a payload vector comprises a packaging sequence that is of the same serotype as the adenoviral capsid.

[0219] In some embodiments, the packaging sequence is an Ad5 packaging sequence and the adenoviral capsid is an Ad5 / 35 or Ad5 / 35++ adenoviral capsid or an Ad5 / 3, Ad5 / 7, Ad5 / 11, Ad5 / 14, Ad5 / 16, Ad5 / 21, Ad5 / 34, Ad5 / 50 or Ad5 / 55 adenoviral capsid. In some embodiments, a payload vector comprises an Ad5 packaging sequence. In some embodiments, an Ad5 packaging sequence comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 5. In some embodiments, an Ad5 packaging sequence comprises a sequence of SEQ ID NO: 5.

[0220] In some embodiments, the packaging sequence is an Ad6 packaging sequence and the adenoviral capsid is an Ad6 / 35 or Ad6 / 35++ adenoviral capsid. In some embodiments, a payload vector comprises an Ad6 packaging sequence. In some embodiments, an Ad6 packaging sequence comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ormore identical to SEQ ID NO: 25. In some embodiments, an Ad6 packaging sequence comprises a sequence of SEQ ID NO: 25.

[0221] In some embodiments, the packaging sequence is an Ad35 packaging sequence and the adenoviral capsid is an Ad35 or Ad35++ adenoviral capsid. In some embodiments, a payload vector comprises an Ad35 packaging sequence. In some embodiments, an Ad35 packaging sequence comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 44. In some embodiments, an Ad35 packaging sequence comprises a sequence of SEQ ID NO: 44.

[0222] Those skilled in the art will be able to select suitable packaging sequences from the genomes of Ad5 and other adenoviral serotypes, e.g., from the genomes of Ad5, Ad6, and Ad35, e.g., by reference to GenBank Accession No. AC_000008.1 (Ad5), GenBank Accession No. OP871032.1 (Ad6), and GenBank Accession No. AC_000019.1 (Ad35). Table 9: Exemplary Packaging SequencesPayload cassettes and therapeutic sequences

[0223] Payload vectors can comprise one or more payload cassettes, which comprise one or more therapeutic sequences for expression in target cells. In some embodiments, a payload vector comprises one or more payload cassettes. In some embodiments, a payload vector comprises a genome that comprises one or more payload cassettes.

[0224] In some embodiments, a payload cassette is engineered in order to achieve one or more desired results such as one or more desired therapeutic effects in a host cell or system, e.g., expression of a protein of therapeutic interest or of expression of a gene editing system, e.g., a CRISPR / Cas editing system, zinc finger nuclease (ZFN) editing system, transcription activator-like effector nucleases (TALEN) editing system, meganuclease editing system, base editing system, prime editing system, epigenetic editing system, RNA editing system, and / or transposase-assisted target-site integration system to generate a sequence modification in the genome of a cell or cells in a subject.

[0225] In some embodiments, a payload cassette comprises a gene. A gene can include not only coding sequences but also regulatory regions such as promoters, enhancers, termination regions, locus control regions (LCRs), termination and polyadenylation signal elements, splicing signal elements, silencers, insulators, and the like. A gene can include introns and other DNA sequences spliced from an expressed mRNA transcript, along with variants resulting from alternative splice sites. Coding sequences can also include alternative synonymous codon usage as compared to a reference sequence, e.g., codon usage modified as compared to a reference in accordance with codon preference of a specific organism or target cell type.

[0226] In some embodiments, a payload cassette can include a single therapeutic sequence or multiple therapeutic sequences. In some embodiments, a payload cassette can include a single coding sequence or a plurality of coding sequences. In some embodiments, a payload cassette can include a single regulatory sequence or a plurality of regulatory sequences. In some embodiments, a payload cassette can include a plurality of coding sequences where the individual expression products of the coding sequences function together, e.g., as in the case of an endonuclease and a guide RNA, or independently, e.g., as two separate proteins that do not directly or indirectly bind. As will be appreciated by those of skill in the art, any payload cassette or payload cassette component (e.g., a therapeutic sequence or regulatory sequence) that is not encoded by a reference wild-type adenoviral genome, e.g., an Ad5 genome, an Ad6 genome, an Ad35 genome, can be referred to herein as a heterologous expression product.

[0227] In some embodiments, a payload cassette of a payload vector, e.g., an adenoviral payload vector, e.g., a HDAd payload vector, or a payload vector genome, e.g., an adenoviral payload vectorgenome, e.g., a HDAd payload vector genome, of the present disclosure can include one or more therapeutic sequences that encode any of a variety of expression products. Exemplary expression products include proteins, including without limitation replacement therapy proteins for treatment of diseases or conditions characterized by low expression or activity of a biologically active protein as compared to a reference level. Exemplary expression products include CRISPR / Cas, base editor, and prime editor systems. Exemplary expression products include antibodies, CARs, and TCRs. Exemplary expression products include small RNAs. In some embodiments, integration of all or a portion of a payload vector genome and / or a payload cassette into a host cell genome is not required in order for delivery to the target cell of a payload vector or genome to produce an intended or target effect, e.g., in certain instances in which the intended or target effect includes editing of the host cell genome by a CRISPR, base editor, or prime editor system. In some embodiments, integration of all or a portion of a payload vector genome and / or a payload cassette is required or preferred in order for delivery to a target cell to produce an intended or target effect, e.g., where expression of a therapeutic sequence is desired in progeny cells of a transduced target cell. In some embodiments, a payload vector genome can include a nucleic acid sequence engineered for integration into a host cell genome (an “integration element”), e.g., by recombination and / or transposition.

[0228] A therapeutic sequence encoding one or more therapeutic polypeptides and / or therapeutic nucleic acids can be readily prepared by synthetic or recombinant methods from the relevant nucleic acid sequence and / or amino acid sequence. In some embodiments, a therapeutic sequence encoding any such therapeutic nucleic acids and / or therapeutic polypeptides can also have one or more restriction enzyme sites at the 5' and / or 3' ends of the coding sequence in order to provide for easy excision and replacement of the therapeutic sequence encoding the sequence with another therapeutic sequence encoding a different sequence. In some embodiments, a therapeutic sequence encoding one or more therapeutic nucleic acids and / or therapeutic polypeptides can be codon optimized for expression in mammalian cells.

[0229] In some embodiments, a therapeutic sequence comprises or is a sequence encoding a therapeutic polypeptide and / or therapeutic nucleic acid. In some embodiments, a therapeutic sequence comprises or is a sequence encoding a therapeutic polypeptide. In some embodiments, a therapeutic sequence comprises or is a sequence encoding a therapeutic nucleic acid.

[0230] For example, a therapeutic sequence may comprise a sequence encoding γ-globin, Factor VIII, γC, JAK3, IL7RA, RAG1, RAG2, DCLRE1C, PRKDC, LIG4, NHEJ1, CD3D, CD3E, CD3Z, CD3G, PTPRC, ZAP70, LCK, AK2, ADA, PNP, WHN, CHD7, ORAI1, STIM1, CORO1A, CIITA, RFXANK, RFX5, RFXAP, RMRP, DKC1, TERT, TINF2, DCLRE1B, SLC46A1, a FANC family nucleic acid sequence / polypeptide / protein (e.g., FancA, FancB, FancC, FancD1 (BRCA2), FancD2, FancE, FancF, FancG, FancI, FancJ (BRIP1), FancL, FancM, FancN (PALB2), FancO (RAD51C), FancP (SLX4), FancQ (ERCC4), FancR (RAD51), FancS (BRCA1), FancT (UBE2T), FancU (XRCC2), FancV (MAD2L2), andFancW (RFWD3)), soluble CD40, CTLA, FasL, an antibody (e.g., that specifically binds CD4, CD5, CD7, CD52, IL1, IL2, IL6, TNF, P53, PTPN22, or DRB1*1501 / DQB1*0602), an antibody to TCR specifically present on autoreactive T cells, IL4, IL10, IL12, IL13, IL1Ra, sIL1RI, sIL1RII, sTNFRI, sTNFRII, globin family polypeptides, WAS, phox, dystrophin, pyruvate kinase, CLN3, ABCD1, arylsulfatase A, SFTPB, SFTPC, NLX2.1, ABCA3, GATA1, ribosomal proteins, TERT, TERC, DKC1, TINF2, CFTR, LRRK2, PARK2, PARK7, PINK1, SNCA, PSEN1, PSEN2, APP, SOD1, TDP43, FUS, ubiquilin 2, C9ORF72, and other therapeutic sequences, therapeutic nucleic acids, therapeutic polypeptides, and / or expression products described herein.

[0231] A therapeutic sequence can be selected to provide a therapeutically effective response against diseases related to red blood cells and clotting. In some embodiments, a disease is a hemoglobinopathy like thalassemia, or a sickle cell disease / trait. A therapeutic gene may be, for example, a gene that induces or increases production of hemoglobin; induces or increases production of β-globin, γ-globin, or α-globin; or increases the availability of oxygen to cells in the body. A therapeutic gene may be, for example, HBB or CYB5R3. A therapeutic gene may be, for example, a gene editing system, e.g., any such gene editing system disclosed herein (e.g., a CRISPR-associated RNA-guided endonuclease, a CRISPR-associated RNA-guided DNA endonuclease inactivated protein (dCas), a base editor), to modify a nucleic acid sequence that encodes γ-globin, e.g., to increase expression of γ-globin. Exemplary effective treatments may, for example, increase blood cell counts, improve blood cell function, or increase oxygenation of cells in patients. In some embodiments, a disease is hemophilia. A therapeutic gene may be, for example, a gene that increases the production of coagulation / clotting factor VIII or coagulation / clotting factor IX, causes production of normal versions of coagulation factor VIII or coagulation factor IX, a gene that reduces production of antibodies to coagulation / clotting factor VIII or coagulation / clotting factor IX, or a gene that causes proper formation of blood clots. Exemplary therapeutic sequences include sequence encoding F8 and F9. Exemplary effective treatments may, for example, increase or induce the production of coagulation / clotting factors VIII and IX; improve functioning of coagulation / clotting factors VIII and IX, or reduce clotting time in subjects.

[0232] In some embodiments of the present disclosure, a payload vector, payload vector genome, and / or payload cassette comprises a therapeutic sequence encoding a globin protein, wherein the globin protein is selected from a γ-globin, a β-globin, and / or an α-globin. As those of skill in the art will appreciate, each of γ-globin, β-globin, and / or α-globin is a component of fetal and / or adult hemoglobin and is therefore useful in various vectors disclosed herein.

[0233] The following references describe particular exemplary sequences of functional globin sequences. References 1-4 relate to α-type globin sequences and references 4-12 relate to β-type globin sequences (including β and γ globin sequences), which sequences are hereby incorporated by reference: (1)GenBank Accession No. Z84721 (Mar. 19, 1997); (2) GenBank Accession No. NM_000517 (Oct. 31, 2000); (3) Hardison et al., J. Mol. Biol. (1991) 222(2):233-249; (4) A Syllabus of Human Hemoglobin Variants (1996), by Titus et al., published by The Sickle Cell Anemia Foundation in Augusta, Ga. (available online at globin.cse.psu.edu); (5) GenBank Accession No. J00179 (Aug.26, 1993) or U01317.1; (6) Tagle et al., Genomics (1992) 13(3):741-760; (7) Grovsfeld et al., Cell (1987) 51(6):975-985; (8) Li et al., Blood (1999) 93(7):2208-2216; (9) Gorman et al., J. Biol. Chem. (2000) 275(46):35914-35919; (10) Slightom et al., Cell (1980) 21(3):627-638; (11) Fritsch et al., Cell (1980) 19(4): 959-972; (12) Marotta et al., J. Biol. Chem. (1977) 252(14):5040-5053. For additional coding and non-coding regions of genes encoding globins see, for example, by Marotta et al., Prog. Nucleic Acid Res. Mol. Biol.19, 165-175, 1976, Lawn et al., Cell 21 (3), 647-651, 1980, and Sadelain et al., PNAS.; 92:6728-6732, 1995. In some embodiments, a globin sequence encodes a G16D gamma globin variant. An exemplary amino acid sequence of hemoglobin subunit β is provided, for example, at NCBI Accession No. P68871. An exemplary amino acid sequence for β-globin is provided, for example, at NCBI Accession No. NP_000509.

[0234] In some embodiments, a therapeutic sequence can encode a therapeutic molecule, such as a checkpoint inhibitor agent, a chimeric antigen receptor molecule specific to one or more cancer antigens, and / or T-cell receptors specific to one or more cancer antigens.

[0235] As another example, in some embodiments, a therapeutic sequence can be selected to provide a therapeutically effective response against a lysosomal storage disorder. In some embodiments, the lysosomal storage disorder is mucopolysaccharidosis (MPS), type I; MPS II or Hunter Syndrome; MPS III or Sanfilippo syndrome; MPS IV or Morquio syndrome; MPS V; MPS VI or Maroteaux-Lamy syndrome; MPS VII or sly syndrome; α-mannosidosis; β-mannosidosis; glycogen storage disease type I, also known as GSDI, von Gierke disease, or Tay Sachs; Pompe disease; Gaucher disease; or Fabry disease. The therapeutic sequence may be, for example, a sequence encoding or inducing production of an enzyme, or that otherwise causes degradation of mucopolysaccharides in lysosomes. Exemplary therapeutic sequences include those of IDUA or iduronidase, IDS, GNS, HGSNAT, SGSH, NAGLU, GUSB, GALNS, GLB1, ARSB, and HYAL1. Exemplary effective genetic therapies for lysosomal storage disorders may, for example, encode or induce the production of enzymes responsible for the degradation of various substances in lysosomes; reduce, eliminate, prevent, or delay the swelling in various organs, including the head (e.g.., Macrocephaly), the liver, spleen, tongue, or vocal cords; reduce fluid in the brain; reduce heart valve abnormalities; prevent or dilate narrowing airways and prevent related upper respiratory conditions like infections and sleep apnea; reduce, eliminate, prevent, or delay the destruction of neurons, and / or the associated symptoms.

[0236] As another example, in some embodiments, a therapeutic sequence can be selected to provide a therapeutically effective response against a hyperproliferative disease. In some embodiments, ahyperproliferative disease is cancer. A therapeutic sequence may encode, for example, a tumor suppressor, an inducer of apoptosis, an enzyme, an antibody, or a hormone. Exemplary therapeutic sequences, and therapeutic products include, but are not limited to, those products that can ameliorate a hyperproliferative effect associated with overexpression, underexpression, mutation (including, but not limited to, point mutation, deletion, truncation, frameshift, splice variants, or insertion), as applicable, for a gene associated with the cause of or exacerbation of a hyperproliferative disease. Such a gene includes, but is not limited to, any one of the following genes selected from the group consisting of (in addition to those listed elsewhere) 101F6, 123F2 (RASSF1), 53BP2, abl, ABLI, ADP, aFGF, APC, ApoAI, ApoAIV, ApoE, ATM, BAI-1, BCMA, BDNF, Beta*(BLU), bFGF, BLC1, BLC6, BRCA1, BRCA2, B7-H3, B7-H4, B7-H6, CALRmut, CBFA1, CBL, C-CAM, CCR2, CD1a, CD3, CD5, CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD70, CD79a, CD79b, CD123, CD137-FAP, CD228, CDH3, CDH6, CEACAM5, CLDN18.2, CLDN6, cMet, CNTF, COX-1, CSFIR, CTS-1, cytosine deaminase, DBCCR-1, DCC, DLL3, Dp, DPC-4, E1A, E2F, EBRB2, EGFR, ENPP3, erb, ERBA, ERBB1, ERBB2 (HER2), ERBB3, ERBB4, ETS1, ETS2, ETV6, Fab, FAP, FCC, FcRH5, FGF, FGFR2b, FGR, FHIT, FLT3, fms, FOX, FRα, Fucosyl GM1, FUS1, FYN, G-CSF, GDAIF, Gene 21 (NPRL2), Gene 26 (CACNA2D2), GM-CSF, GMF, GPC3, GPRC5D, GRPR, gsp, HCR, HER2, HER3, HIC-1, hK2, HRAS, hst, IGF, IL-1, IL-2, IL-3, IL-5, IL-6, IL-7, IL-8, IL- 9, IL-11, ING1, Integrin beta-3 / 5, Integrin beta-6, interferon α, interferon β, interferon γ, IRF-1, JUN, KRAS, KRAS G12D, LIF, LUCA-1 (HYAL1), LUCA-2 (HYAL2), LYN, MADH4, MADR2, MAGEA4- MAGEA8, MCC, mda7, MDM2, MEN-I, MEN-II, Mesothelin, MET, MLL, MLL1, MMAC1, MYB, MYC, MYCL1, MYCN, Nectin-4, Neoantigen, neu, NF-1, NF-2, NGF, NOEY1, NOEY2, NRAS, NT3, NT5, OVCA1, p16, p21, p27, p57, p73, p300, PGS, PIM1, PL6, PML, PSCA, PSMA, PTEN, PVRIG, raf, Rap1A, ras, Rb, RB1, RET, rks-3, ROR1, ScFv, scFV ras, SEM A3, SEZ6, SLAMF7 / CS1, SRC, SSTR, SSTR2, STEAP1, STEAP2, TALI, TCL3, TFPI, thrombospondin, thymidine kinase, Tissue factor (TF), TNF, TP53, TP53 R175H, TRBC1, trk, TROP2, T-VEC, VEGF, VHL, WT1, WT-1, YES, and zac1. In some embodiments, a gene associated with the cause of or exacerbation of a hyperproliferative disease is associated with a solid tumor. In some embodiments, a gene associated with the cause of or exacerbation of a hyperproliferative disease is selected from B7-H3, B7-H4, B7-H6, CALRmut, CCR2, CD30, CD70, CD137-FAP, CD228, CDH3, CDH6, CEACAM5, CLDN18.2, CLDN6, cMet, DLL3, EGFR, ENPP3, FAP, FcRH5, FGFR2b, FRα, Fucosyl GM1, GPC3, GRPR, HER2, HER3, hK2, Integrin beta-3 / 5, Integrin beta- 6, KRAS, KRAS G12D, LIF, MAGEA4-MAGEA8, Mesothelin, MET, MLL1, Nectin-4, Neoantigen, PSMA, PSCA, PVRIG, ROR1, SEZ6, SSTR, SSTR2, STEAP1, STEAP2, Tissue factor (TF), TP53, TP53 R175H, TROP2, and VEGF. In some embodiments, a gene associated with the cause of or exacerbation of a hyperproliferative disease is associated with a liquid tumor. In some embodiments, a gene associated with the cause of or exacerbation of a hyperproliferative diseases is selected from BCMA, CD1a, CD3,CD5, CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD79a, CD79b, CD123, FLT3, GPRC5D, SLAMF7 / CS1, and TRBC1. Exemplary effective gene therapies may suppress or eliminate tumors, result in a decreased number of cancer cells, reduced tumor size, slow or eliminate tumor growth, or alleviate symptoms caused by tumors.

[0237] As another example, in some embodiments, a therapeutic sequence can be selected to provide a therapeutically effective response against an infectious disease. In some embodiments, an infectious disease is human immunodeficiency virus (HIV). A therapeutic sequence may be, for example, a sequence encoding a therapeutic nucleic acid or therapeutic polypeptide rendering immune cells resistant to HIV infection, or which enables immune cells to effectively neutralize the virus via immune reconstruction, polymorphisms of genes encoding proteins expressed by immune cells; a therapeutic nucleic acid or therapeutic polypeptide advantageous for fighting infection that is not expressed in a subject; an infectious agent, receptor or coreceptor; ligands for receptors or coreceptors; a therapeutic nucleic acid or therapeutic polypeptide essential for viral replication including ribozymes, antisense RNA, small interfering RNA (siRNA) or decoy RNA to block actions of certain transcription factors; dominant negative viral proteins, intracellular antibodies, intrakines and suicide genes. Exemplary therapeutic sequences, and therapeutic products encoded thereby, include α2β1; αvβ3; αvβ5; αvβ63; BOB / GPR15; Bonzo / STRL-33 / TYMSTR; CCR2; CCR3; CCR5; CCR8; CD4; CD46; CD55; CXCR4; aminopeptidase-N; HHV-7; ICAM; ICAM-1; PRR2 / HveB; HveA; α-dystroglycan; LDLR / α2MR / LRP; PVR; PRR1 / HveC; and laminin receptor. A therapeutically effective amount for treatment of HIV, for example, may increase immunity of a subject against HIV, ameliorate a symptom associated with AIDS or HIV, or induce an innate or adaptive immune response in a subject against HIV. An immune response against HIV may include antibody production and result in prevention of AIDS and / or ameliorate a symptom of AIDS or HIV infection exhibited by a subject, or decrease or eliminate HIV infectivity and / or virulence.

[0238] In some embodiments, a therapeutic sequence encodes a therapeutic polypeptide comprising any of a variety of binding domains. For example, in some embodiments, a therapeutic polypeptide comprises or is an antibody or antigen-binding fragment thereof, a chimeric antigen receptor (CAR), a T- cell receptor (TCR), or other binding polypeptides.

[0239] In some embodiments, an antibody is polyclonal, monoclonal, monospecific, or multispecific antibodies (including bispecific antibodies). In some embodiments, an antibody includes at least one light chain monomer or dimer, at least one heavy chain monomer or dimer, at least one heavy chain-light chain dimer, or a tetramer that includes two heavy chain monomers and two light chain monomers. Moreover, the term “antibody” can include (unless otherwise stated or clear from context) any art-known constructs or formats utilizing antibody structural and / or functional features including, without limitation, intrabodies, domain antibodies, antibody mimetics, Zybodies®, Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’fragments, Fd fragments, isolated CDRs or sets thereof, single chain antibodies, single-chain Fvs (scFvs), disulfide-linked Fvs (sdFv), polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), cameloid antibodies, camelized antibodies, masked antibodies (e.g., Probodies®), affybodies, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), Small Modular ImmunoPharmaceuticals (“SMIPsTM”), single chain or Tandem diabodies (TandAb®), VHHs, Anticalins®, Nanobodies® minibodies, BiTE®s, ankyrin repeat proteins or DARPINs®, Avimers®, DARTs, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, and KALBITOR®s, CARs, engineered TCRs, and antigen-binding fragments of any of the above.

[0240] In some embodiments, an antibody or antigen-binding fragment thereof, or a therapeutic polypeptide comprising a binding domain, can bind to a cell surface protein or a secreted protein. In some further embodiment, the cell surface protein or secreted protein is a protein selected from the group consisting of AFP, ALPP, PD-1, PD-L1, LAG-3, TIM-3, BAFFR, B7-H3 (CD276), B7-H4, B7-H6, CALRmut, CCR2, CD1a, CD3, CD4, CD5, CD7, CD19, CD20, CD22, CD30, CD33, CD34, CD37, CD38, CD44v6, CD52, CD56, CD70, CD73, CD79a, CD79b, CD80, CD110, CD117, CD123, CD124, CD126, CD133, CD137-FAP, CD138, CD147, CD171, CD228, CD269, CD276, CDH3, CDH6, C7R, Chlorotoxin, GLY, GUCY2C, IL1, IL2, IL6, a TCR specifically present on autoreactive T cells, IL4, IL10, IL12, IL13, IL13Rα2, IL1Ra, IL1RAP, sIL1RI, sIL1RII, ILT3, TNF, ABCA3, ABCD1, ADA, AK2, APP, arginase, arylsulfatase A, AXL, A1AT, BCMA, CCCR, CD3D, CD3E, CD3G, CAIX, CD3Z, CEA, CEACAM5, CFTR, CHD7, CIITA, CLDN, CLDN6, CLDN18.2, CLL1, CLN3, complement factor, CORO1A, CTLA, C1 inhibitor, C9ORF72, c-Met, DLL3, DCLRE1B, DCLRE1C, decoy receptors, DKC1, DRB1*1501 / DQB1*0602, DR5, dystrophin, enzymes, EGFR, EGFRvIII, ENPP3, EpCam, EphA2, Factor VIII, FANC polypeptides (FancA, FancB, FancC, FancD1 (BRCA2), FancD2, FancE, FancF, FancG, FancI, FancJ (BRIP1), FancL, FancM, FancN (PALB2), FancO (RAD51C), FancP (SLX4), FancQ (ERCC4), FancR (RAD51), FancS (BRCA1), FancT (UBE2T), FancU (XRCC2), FancV (MAD2L2), and FancW (RFWD3)), FAP, FasL, FcRH5, FGFR2b FLT3, FUS, Fucosyl GM1, GATA1, GFRalpha4, gp100, globin polypeptides (i.e., γ-globin), F8, GD2, glutaminase, GPC3, GPRC5D, GRPR, FRalpha, HBA1, HBA2, HBB, HER1, HER2, HER3, HER4, hK2, ICAM-1, IL7RA, Integrin beta-3 / 5, Integrin beta-6, JAK3, KLK2, KRAS, KRAS G12D, LCK, LeY, LIF, LIG4, LMP1, LRRK2, MAGEA4-MAGEA8, Mesothelin, MET, MLL1, MUC1, MUC16, MUC17, MSLN, Nectin-4, Neoantigen, NKG2D, NKG2DL, NKR2, NHEJ1, NLX2.1, NY-ESO-1, ORAI1, PARK2, PARK7, phox, PINK1, PI3K, PNP, PRKDC, PSCA, PSEN1, PSEN2, PSMA, PTPN22, PTPRC, PVRIG, P53, pyruvate kinase, RAG1, RAG2, RFXANK, RFXAP, RFX5, RMRP, ribosomal proteins, ROR1, ROR2, SEZ6, SLAMF7, SFTPB, SFTPC, SOD1, soluble CD40, SSTR, SSTR2, STEAP1, STEAP2, STIM1, sTNFRI, sTNFRII, SLAMF7 / CS1, SLC46A1,SNCA, TDP43, TERT, TERC, TINF2, Tissue factor (TF), TM451F1, TnMUC1, TP53, TP53 R175H, TSLPR, TRBC1, TROP, TROP2, ubiquilin 2, VEGF, VEGFR2, WAS, WHN, ZAP70, γC, and other polypeptides described herein. In some embodiments, an antibody or antigen-binding fragment thereof, or a therapeutic polypeptide comprising a binding domain, can bind to a protein associated with a solid tumor. In some embodiments, an antibody or antigen-binding fragment thereof, or a therapeutic polypeptide comprising a binding domain, can bind to a protein selected from the group consisting of B7-H3, B7-H4, B7-H6, CALRmut, CCR2, CD30, CD70, CD137-FAP, CD228, CDH3, CDH6, CEACAM5, CLDN18.2, CLDN6, cMet, DLL3, EGFR, ENPP3, FAP, FcRH5, FGFR2b, FRα, Fucosyl GM1, GPC3, GRPR, HER2, HER3, hK2, Integrin beta-3 / 5, Integrin beta-6, KRAS, KRAS G12D, LIF, MAGEA4-MAGEA8, Mesothelin, MET, MLL1, Nectin-4, Neoantigen, PSMA, PSCA, PVRIG, ROR1, SEZ6, SSTR, SSTR2, STEAP1, STEAP2, Tissue factor (TF), TP53, TP53 R175H, TROP2, and VEGF. In some embodiments, an antibody or antigen-binding fragment thereof, or a therapeutic polypeptide comprising a binding domain, can bind to a protein associated with a liquid tumor. In some embodiments, an antibody or antigen-binding fragment thereof, or a therapeutic polypeptide comprising a binding domain, can bind to a protein from the group consisting of BCMA, CD1a, CD3, CD5, CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD79a, CD79b, CD123, FLT3, GPRC5D, SLAMF7 / CS1, and TRBC1.

[0241] In some embodiments, a therapeutic polypeptide comprises or is a CAR. CARs can include several distinct subcomponents that can cause cells to recognize and kill target cells such as cancer cells. Subcomponents include at least an extracellular component and an intracellular component.

[0242] An extracellular CAR component can include a binding domain that specifically binds a marker that is preferentially present on the surface of unwanted cells. In some embodiments, an extracellular CAR component can include a binding domain that specifically binds to a cell surface protein mentioned herein, e.g., without limitation B7-H3, B7-H4, B7-H6, CALRmut, CCR2, CD1a, CD3, CD5, CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD70, CD79a, CD79b, CD123, CD137-FAP, CD228, CDH3, CDH6, CEACAM5, CLDN18.2, CLDN6, cMet, DLL3, EGFR, ENPP3, FAP, FcRH5, FGFR2b, FLT3, FRα, Fucosyl GM1, GPC3, GPRC5D, GRPR, HER2, HER3, hK2, Integrin beta-3 / 5, Integrin beta-6, KRAS, KRAS G12D, LIF, MAGEA4-MAGEA8, Mesothelin, MET, MLL1, Nectin-4, Neoantigen, PSMA, PSCA, PVRIG, ROR1, SEZ6, SLAMF7 / CS1, SSTR, SSTR2, STEAP1, STEAP2, Tissue factor (TF), TP53, TP53 R175H, TRBC1, TROP2, VEGF, or BCMA, etc. In some embodiments, an extracellular CAR component can include a binding domain that specifically binds to a protein associated with a solid tumor. In some embodiments, an extracellular CAR component can include a binding domain that specifically binds to B7- H3, B7-H4, B7-H6, CALRmut, CCR2, CD30, CD70, CD137-FAP, CD228, CDH3, CDH6, CEACAM5, CLDN18.2, CLDN6, cMet, DLL3, EGFR, ENPP3, FAP, FcRH5, FGFR2b, FRα, Fucosyl GM1, GPC3, GRPR, HER2, HER3, hK2, Integrin beta-3 / 5, Integrin beta-6, KRAS, KRAS G12D, LIF, MAGEA4-MAGEA8, Mesothelin, MET, MLL1, Nectin-4, Neoantigen, PSMA, PSCA, PVRIG, ROR1, SEZ6, SSTR, SSTR2, STEAP1, STEAP2, Tissue factor (TF), TP53, TP53 R175H, TROP2, or VEGF. In some embodiments, an extracellular CAR component can include a binding domain that specifically binds to a protein associated with a liquid tumor. In some embodiments, an extracellular CAR component can include a binding domain that specifically binds to BCMA, CD1a, CD3, CD5, CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD79a, CD79b, CD123, FLT3, GPRC5D, SLAMF7 / CS1, or TRBC1. When a binding domain binds such markers, the intracellular component directs a cell to destroy the bound cancer cell. A binding domain is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other formats which include an antibody-like antigen binding site.

[0243] Intracellular CAR components provide activation signals based on inclusion of an effector domain. First generation CARs have utilized a cytoplasmic region of CD3ζ as an effector domain. Second generation CARs have utilized CD3ζ in combination with cluster of differentiation 28 (CD28) or 4-1BB (CD137), while third generation CARs have utilized CD3ζ in combination with CD28 and 401BB within intracellular effector domains.

[0244] Intracellular or otherwise cytoplasmic signaling components of a CAR are responsible for activation of the cell in which the CAR is expressed. The term “intracellular signaling components” or “intracellular components” is thus meant to include any portion of the intracellular domain sufficient to transduce an activation signal. Intracellular components of expressed CAR can include effector domains. An effector domain is an intracellular portion of a fusion protein or receptor that can directly or indirectly promote a biological or physiological response in a cell when receiving the appropriate signal. In some embodiments, an effector domain is part of a protein or protein complex that receives a signal when bound, or it binds directly to a target molecule, which triggers a signal from the effector domain. An effector domain may directly promote a cellular response when it contains one or more signaling domains or motifs, such as an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, an effector domain will indirectly promote a cellular response by associating with one or more other proteins that directly promote a cellular response, such as co-stimulatory domains.

[0245] Effector domains can provide for activation of at least one function of a modified cell upon binding to a cellular marker expressed by a cancer cell. Activation of a modified cell can include one or more of differentiation, proliferation and / or activation or other effector functions. In some embodiments, an effector domain can include an intracellular signaling component including a T cell receptor and a co- stimulatory domain which can include a cytoplasmic sequence from a co-receptor or co-stimulatory molecule.

[0246] An effector domain can include one, two, three or more receptor signaling domains, intracellular signaling components (e.g., cytoplasmic signaling sequences), co-stimulatory domains, orcombinations thereof. Exemplary effector domains include signaling and stimulatory domains selected from: 4-1BB (CD137), CARD11, CD3γ, CD3δ, CD3ε, CD3ζ, CD27, CD28, CD79A, CD79B, DAP10, FcRα, FcRβ (FcεR1b), FcRγ, Fyn, HVEM (LIGHTR), ICOS, LAG3, LAT, Lck, LRP, NKG2D, NOTCH1, pTα, PTCH2, OX40, ROR2, Ryk, SLAMF1, Slp76, TCRα, TCRβ, TRIM, Wnt, Zap70, or any combination thereof. In some embodiments, exemplary effector domains include signaling and co-stimulatory domains selected from: CD86, FcγRIIa, DAP12, CD30, CD40, PD-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, GADS, PAG / Cbp, NKp44, NKp30, or NKp46.

[0247] Intracellular signaling component sequences that act in a stimulatory manner may include ITAMs. Examples of ITAMs including primary cytoplasmic signaling sequences include those derived from CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD66d, CD79a, CD79b, and common FcRγ (FCER1G), FcγRlla, FcRβ (Fcε Rib), DAP10, and DAP12. In some embodiments, variants of CD3ζ retain at least one, two, three, or all ITAM regions.

[0248] In some embodiments, an effector domain includes a cytoplasmic portion that associates with a cytoplasmic signaling protein, where the cytoplasmic signaling protein is a lymphocyte receptor or signaling domain thereof, a protein including a plurality of ITAMs, a co-stimulatory domain, or any combination thereof.

[0249] Additional examples of intracellular signaling components include cytoplasmic sequences of a CD3ζ chain, and / or co- receptors that act in concert to initiate signal transduction following binding domain engagement.

[0250] A co-stimulatory domain is domain whose activation can be required for an efficient lymphocyte response to cellular marker binding. Some molecules are interchangeable as intracellular signaling components or co-stimulatory domains. Examples of costimulatory domains include CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83. For example, CD27 co-stimulation has been demonstrated to enhance expansion, effector function, and survival of human CART cells in vitro and augments human T cell persistence and anti-cancer activity in vivo (Song et al. Blood. 2012; 119(3):696-706). Further examples of such co-stimulatory domain molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46,CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11lc, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, and CD19a.

[0251] In some embodiments, an amino acid sequence of the intracellular signaling component includes a variant of CD3ζ and a portion of the 4-1BB intracellular signaling component.

[0252] In some embodiments, an intracellular signaling component includes (i) all or a portion of a signaling domain of CD3ζ, (ii) all or a portion of the signaling domain of 4-1BB, or (iii) all or a portion of a signaling domain of CD3ζ and 4-1BB.

[0253] Intracellular components may also include one or more of a protein of a Wnt signaling pathway (e.g., LRP, Ryk, or ROR2), NOTCH signaling pathway (e.g., NOTCH1, NOTCH2, NOTCH3, or NOTCH4), Hedgehog signaling pathway (e.g., PTCH or SMO), receptor tyrosine kinases (RTKs) (e.g., epidermal growth factor (EGF) receptor family, fibroblast growth factor (FGF) receptor family, hepatocyte growth factor (HGF) receptor family, insulin receptor (IR) family, platelet-derived growth factor (PDGF) receptor family, vascular endothelial growth factor (VEGF) receptor family, tropomycin receptor kinase (Trk) receptor family, ephrin (Eph) receptor family, AXL receptor family, leukocyte tyrosine kinase (LTK) receptor family, tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE) receptor family, receptor tyrosine kinase-like orphan (ROR) receptor family, discoidin domain (DDR) receptor family, rearranged during transfection (RET) receptor family, tyrosine-protein kinase-like (PTK7) receptor family, related to receptor tyrosine kinase (RYK) receptor family, or muscle specific kinase (MuSK) receptor family); G-protein-coupled receptors, GPCRs (Frizzled or Smoothened); serine / threonine kinase receptors (BMPR or TGFR); or cytokine receptors (IL1R, IL2R, IL7R, or IL15R).

[0254] CARs generally also include one or more linker sequences that are used for a variety of purposes within the molecule. For example, a transmembrane domain can be used to link an extracellular component of a CAR to an intracellular component. A flexible linker sequence often referred to as a spacer region that is membrane-proximal to a binding domain can be used to create additional distance between a binding domain and a cellular membrane. This additional distance can be beneficial to reduce steric hindrance to binding based on proximity to the membrane. A common spacer region used for this purpose is an IgG4 linker. More compact spacers or longer spacers can be used, depending on the targeted cell marker. Other potential CAR subcomponents are described in more detail elsewhere herein.

[0255] Transmembrane domains within a CAR molecule, often serve to connect the extracellular component and intracellular component through the cell membrane. A transmembrane domain can anchor the expressed molecule in the modified cell’s membrane.

[0256] A transmembrane domain can be derived either from a natural and / or a synthetic source. When a source is natural, a transmembrane domain can be derived from any membrane-bound or transmembrane protein. Transmembrane domains can include at least transmembrane region(s) of an α, β or ζ chain of a T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22; CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In some embodiments, a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD 11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9(CD229), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKG2D, or NKG2C. In some embodiments, a variety of human hinges can be employed as well including the human Ig (immunoglobulin) hinge (e.g., an IgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge or a CD8a hinge.

[0257] In some embodiments, a therapeutic polypeptide comprises or is a TCR. TCRs include naturally occurring T cell receptors. Payloads of the present disclosure can encode a TCR or a CAR / TCR hybrids that includes an element of a TCR and an element of a CAR. For example, a CAR / TCR hybrid could have a naturally occurring TCR binding domain with an effector domain that the TCR binding domain is not naturally associated with. A CAR / TCR hybrid could have a mutated TCR binding domain and an ITAM signaling domain. A CAR / TCR hybrid could have a naturally occurring TCR with an inserted non- naturally occurring spacer region or transmembrane domain.

[0258] In some embodiments, a payload vector comprises two or more payload cassettes in order to achieve two or more desired effects in a host, e.g., expression of two transgenes or genes of interest, expression of a transgene or gene of interest and a gene editing system, expression of gene editing systems to edit at least two different sites. Examples of gene editing systems, include but are not limited to, a CRISPR / Cas editing system, zinc finger nuclease (ZFN) editing system, transcription activator-like effector nucleases (TALEN) editing system, meganuclease editing system, base editing system, prime editing system, epigenetic editing system, RNA editing system, and / or transposase-assisted target-site integration system. Epigenetic editors generally incorporate DNA or Histone methylases, demethylases, acetylases ordeacetylases alone or in combination with transcriptional activation or transcriptional repression domains, depending on the desired utility.

[0259] In some embodiments, a therapeutic sequence encodes at least one component, or all components, of a gene editing system. Gene editing systems of the present disclosure include CRISPR systems, base editing, and prime editing systems. Broadly, gene editing systems can include a plurality of components including a gene editing enzyme selected from a CRISPR-associated RNA-guided endonuclease, a base editing enzyme, and a prime editing enzyme and at least one gRNA. Accordingly, gene editing systems of the present disclosure can include either (i) in the case of a CRISPR system, a CRISPR enzyme that is a CRISPR-associated RNA-guided endonuclease and at least one guide RNA (gRNA), (ii) in the case of a base editing system, a base editing enzyme and at least one gRNA, or (iii) in the case of a prime editing system and at least one prime editing gRNA. Nucleotide sequences encoding gene editing systems as disclosed herein are typically too large for inclusion in many limited-capacity vector systems, but the large capacity of adenoviral vectors permits inclusion of such sequences in adenoviral vectors and genomes of the present disclosure. An additional advantage of adenoviral vectors and genomes with payloads encoding gene editing systems or components of the present disclosure is that adenoviral genomes do not naturally integrate into host cell genomes, which facilitates transient expression of gene editing systems and components, which can be desirable, e.g., to avoid immunogenicity and / or genotoxicity.

[0260] In some embodiments, a gene editing system comprises or is a zing finger nuclease (ZFN). For instance, a ZFN is an artificial endonuclease that consists of a designed zinc finger protein (ZFP) fused to the cleavage domain of the FokI restriction enzyme. A ZFN may be redesigned to cleave new targets by developing ZFPs with new sequence specificities. For genome engineering, a ZFN is targeted to cleave a chosen genomic sequence. A cleavage event induced by the ZFN provokes cellular repair processes that in turn mediate efficient modification of the targeted locus. If a ZFN-induced cleavage event is resolved via non-homologous end joining, this can result in small deletions or insertions, effectively leading to gene knockout. If a break is resolved via a homology-based process in the presence of an investigator-provided donor, small changes or entire transgenes can be transferred, often without selection, into a chromosome; which can be referred to as gene correction and gene addition, respectively.

[0261] In some embodiments, a gene editing system (e.g., a CRISPR system, base editing system, or prime editing system) is engineered to modify a nucleic acid sequence that encodes γ-globin, e.g., to increase expression of γ-globin. The main fetal form of hemoglobin, hemoglobin F (HbF) is formed by pairing of γ-globin polypeptide subunits with α-globin polypeptide subunits. Human fetal γ -globin genes (HBG1 and HBG2; two highly homologous genes produced by evolutionary duplication) are ordinarily silenced around birth, while expression of adult β-globin gene expression (HBB and HBD) increases.Mutations that cause or permit persistent expression of fetal γ-globin throughout life can ameliorate phenotypes of β-globin deficiencies. Thus, reactivation of fetal γ-globin genes can be therapeutically beneficial, particularly in subjects with β-globin deficiency. A variety of mutations that cause increased expression of γ-globin are known in the art (see, e.g., Wienert, Trends in Genetics 34(12): 927-940, 2018, which is incorporated herein by reference in its entirety and with respect to mutations that increase expression of γ-globin). Certain such mutations are found in the HBG1 promoter or HBG2 promoter.

[0262] In some embodiments, a gene editing system designed to increase expression of γ-globin includes an HBG1 / 2 promoter-targeted gRNA that is designed to increase expression of γ-globin coding by modification and / or inactivation of a BCL11A repressor protein binding site. In some embodiments, a gene editing system designed to increase expression of γ-globin includes a bcl11a-targeted gRNA that is designed to increase expression of γ-globin by modification and / or inactivation of the erythroid bcl11a enhancer to reduce BCL11A repressor protein expression in erythroid cells. In some embodiments, a gene editing system designed to increase expression of γ-globin includes a gRNA targeted to cause a loss of function mutation in the gene encoding BCL11A.

[0263] The present disclosure includes, among other things, CRISPR editing agents and systems, and therapeutic sequences encoding the same, e.g., wherein the therapeutic sequence is present in an adenoviral vector or genome. A CRISPR editing system can include a CRISPR editing enzyme and / or at least one gRNA as components thereof. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated protein) nuclease systems are engineered nuclease systems used for genetic engineering that are based on a bacterial system. It is based in part on the adaptive immune response of many bacteria and archaea. When a virus or plasmid invades a bacterium, segments of the invader's DNA are converted into CRISPR RNAs (crRNA) by the bacteria’s “immune” response. A crRNA then associates, through a region of partial complementarity, with another type of RNA called tracrRNA to guide a Cas nuclease to a region homologous to the crRNA in a target DNA called a “protospacer.” A Cas nuclease cleaves the DNA to generate blunt ends at the double-strand break at sites specified by a 20- nucleotide complementary strand sequence contained within a crRNA transcript. In some instances, a Cas nuclease requires both the crRNA and the tracrRNA for site-specific DNA recognition and cleavage. In other instances, a Cas nuclease requires only a crRNA for site-specific DNA recognition and cleavage.

[0264] Guide RNAs (gRNAs) are an example of an element that can target CRISPR editing. In its simplest form, gRNA provides a sequence that targets a site within a genome based on complementarity (e.g., crRNA). As explained below, however, gRNA can also include additional components. For example, in some embodiments, gRNA can include a targeting sequence (e.g., crRNA) and a component to link the targeting sequence to a cutting element. This linking component can be tracrRNA. In some embodiments, gRNA including crRNA and tracrRNA can be expressed as a single molecule referred to as single gRNA(sgRNA). gRNA can also be linked to a cutting element through other mechanisms such as through a nanoparticle or through expression or construction of a dual or multi-purpose molecule. Those of skill in the art will appreciate that gRNA or other targeting elements that can be used to generate a selected nucleic acid sequence correction or modification, e.g., in a host cell of an adenoviral payload vector or genome of the present disclosure, can be readily designed and implemented, e.g., based on available sequence information.

[0265] Cas9 CRISPR / Cas systems will typically use a gRNA that includes a crRNA and a tracrRNA, optionally expressed as a single molecule referred to as single gRNA (sgRNA). Other CRISPR / Cas systems, such as Cas12 or Cas13-derived systems, use a gRNA that includes a crRNA but lacks a tracrRNA. In some embodiments, the crRNA includes at least a fragment that base pairs with a complementary target nucleic acid (e.g., at least 80% identity between the fragment and the complement of the target nucleic acid, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity), where the fragment can be 10 to 40 nucleotides in length (e.g., equal to or about 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides in length, e.g., 17-24 or 17-20 nucleotides in length), e.g., where the target sequence is relative to an appropriate PAM site. In some embodiments, a crRNA includes a sequence that forms a stemloop structure and binds with and / or recruits a Cas protein, optionally fused to other domains.

[0266] Examples of cutting elements include nucleases. CRISPR-Cas loci have more than 50 gene families and there are no strictly universal genes, indicating fast evolution and extreme diversity of loci architecture. Exemplary Cas nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12; including, e.g., spCas9, dCas9, nCas9, and Cas9-SpRY), Cas9d, Cas10, Cas12 (e.g., Cas12a (e.g., LbCas12a, AsCas12a, FnCas12a, MB3Cas12a, Cas12a-M11, Cas12a- M13 (e.g., Cas12a-M13-1), Cas12a-M26 (e.g., Cas12a-M26-1), Cas12a-M28 (e.g., Cas12a-M28-1), Cas12a-M29 (e.g., Cas12a-M29-1), Cas12a-M30 (e.g., Cas12a-M30-1), Cas12a-M31 (e.g., Cas12a-M31- 1), Cas12a-M32 (e.g., Cas12a-M32-1), Cas12a-M57, Cas12a-M58, Cas12a-M59, Cas12a-M60 (e.g., Cas12a-M60-9), Cas12a-M61, or Cas12a-M62), Cas12b, Cas12c, Cas12g, Cas12h, or Cas12i), Cas13a, Cas13b, Cas13d, Cas14, Cas-Phi, CasX, C2c3, C2c2, C2c1, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and variants thereof.

[0267] There are three main types of Cas nucleases (type I, type II, and type III), and 10 subtypes including 5 type I, 3 type II, and 2 type III proteins (see, e.g., Hochstrasser and Doudna, Trends Biochem Sci, 2015:40(l):58-66). Type II Cas nucleases include Cas1, Cas2, Csn2, and Cas9. These Cas nucleases are known to those skilled in the art. For example, an amino acid sequence of a Streptococcus pyogenes wild-type Cas9 polypeptide is set forth, e.g., in NCBI accession no. NP_269215, and an amino acidsequence of Streptococcus thermophilus wild-type Cas9 polypeptide is set forth, e.g., in NCBI accession no. WP_011681470.

[0268] In some embodiments, Cas9 refers to an RNA-guided double-stranded DNA-binding nuclease protein or nickase protein. Wild-type Cas9 nuclease has two functional domains, e.g., RuvC and HNH, that cut different DNA strands. Cas9 can induce double-strand breaks in genomic DNA (target DNA) when both functional domains are active. A Cas9 enzyme, in some embodiments, includes one or more catalytic domains of a Cas9 protein derived from bacteria such as Corynebacter, Sutterella, Legionella, Treponema, Filif actor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, and Campylobacter. In some embodiments, a Cas9 is a fusion protein, e.g. the two catalytic domains are derived from different bacterial species.

[0269] In some embodiments, crRNA and tracrRNA can be combined into one molecule called a single gRNA (sgRNA). In this engineered approach, the sgRNA guides Cas to target any desired sequence (see, e.g., Jinek et al., Science 337:816-821, 2012; Jinek et al., eLife 2:e00471, 2013; Segal, eLife 2:e00563, 2013). Thus, a CRISPR / Cas system can be engineered to create a double-strand break at a desired target in a genome of a cell, and harness the cell's endogenous mechanisms to repair the induced break by HDR, or NHEJ. Particular embodiments described herein utilize homology arms to promote HDR at defined integration sites.

[0270] In some embodiments, variants of a Cas9 nuclease include a single inactive catalytic domain, such as a RuvC or HNH enzyme or a nickase. A Cas9 nickase has only one active functional domain and, in some embodiments, cuts only one strand of the target DNA, thereby creating a single strand break or nick. In some embodiments, a mutant Cas9 nuclease having at least a D10A mutation is a Cas9 nickase. In some embodiments, a mutant Cas9 nuclease having at least a H840A mutation is a Cas9 nickase. Other examples of mutations present in a Cas9 nickase include N854A and N863A. A double-strand break is introduced using a Cas9 nickase if at least two DNA-targeting RNAs that target opposite DNA strands are used. A double-nicked induced double-strand break is repaired by HDR or NHEJ. This gene editing strategy generally favors HDR and decreases the frequency of indel mutations at off-target DNA sites. A Cas9 nuclease or nickase, in some embodiments, is codon-optimized for the target cell or target organism.

[0271] Any nuclease of a CRISPR system described herein can be inactivated, partially or wholly, and used. In some embodiments, a CRISPR-associated RNA-guided endonuclease is a CRISPR-associated RNA-guided DNA endonuclease inactivated protein (dCas). In some embodiments, a CRISPR-associated RNA-guided DNA endonuclease inactivated protein (dCas) is a dCas12. In some embodiments, a CRISPR- associated RNA-guided DNA endonuclease inactivated protein (dCas) is a dCas12a. In some embodiments, a CRISPR-associated RNA-guided DNA endonuclease inactivated protein (dCas) is a dCas9.

[0272] The present disclosure includes, among other things, base editing agents and systems, and therapeutic sequences encoding the same, e.g., where the therapeutic sequence is present in an adenoviral vector or genome. A base editing system can include a base editing enzyme and / or at least one gRNA as components thereof. A base editing system can utilize a deaminase (e.g., a base editing system) for editing of nucleic acid targets.

[0273] Deamination is the removal of an amine group from a molecule such as a nucleotide of a nucleic acid. Deamination of a nucleotide can cause changes in the sequence of a nucleic acid, and deaminases are useful in editing for at least that reason. Deamination of adenosine (A) yields inosine (I), which has the same base pairing preferences as a guanosine in DNA and is thus recognized by cell replication machinery as guanosine, resulting in an A-T to G-C transition. Deamination of cytosine (C) yields uridine (U), which is recognized by cell replication machinery as thymine, resulting in a C-G to T-A transition. Collectively, cytosine and adenosine deamination can be used to cause transitions from A to G, T to C, C to T, or G to A. Other deaminase activities are also known. For example, deamination of 5-methylcytosine yields thymine and deamination of guanosine yields xanthine, though xanthine, like guanosine, pairs with cytosine. Deaminases that deaminate cytosine can be referred to as cytosine deaminases. Deaminases that deaminate adenosine can be referred to as adenosine deaminases.

[0274] In some embodiments, a base editing enzyme includes a cytidine deaminase domain or an adenine deaminase domain. Certain embodiments utilize a cytidine deaminase domain as the nucleobase deaminase enzyme. Particular embodiments utilize an adenine deaminase domain as the nucleobase deaminase enzyme.

[0275] Examples of cytosine deaminase enzymes (CBEs) include APOBEC1, APOBEC3A, APOBEC3G, CDA1, and AID. APOBEC1 particularly accepts single-stranded (ss)DNA as a substrate but is incapable of acting on double-stranded (ds)DNA.

[0276] For adenosine base editors (ABEs), exemplary adenosine deaminases that can act on DNA for adenine base editing include mutant TadA adenosine deaminases (TadA*) that accepts DNA as its substrate. E. coli TadA typically acts as a homodimer to deaminate adenosine in transfer RNA (tRNA). TadA* deaminase catalyzes the conversion of a target ‘A’ to ‘I’ (inosine), which is treated as ‘G’ by cellular polymerases. Subsequently, an original genomic A-T base pair can be converted to a G-C pair. As the cellular inosine excision repair is not as active as uracil excision, ABE does not require any additional inhibitor protein like UGI in CBE. In some embodiments, an ABE can include one or more, or all, of three components including a wild-type E. coli tRNA-specific adenosine deaminase (TadA) monomer, which can play a structural role during base editing, a TadA* mutant TadA monomer that catalyzes deoxyadenosine deamination, and / or a Cas nickase such as Cas9(D10A). In some embodiments, there is a linker positioned between TadA and TadA*, and in some embodiments there is a linker positioned betweenTadA* and the Cas nickase. In some embodiments, one or both linkers includes at least 6 amino acids, e.g., at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids (e.g., having a lower bound of 5, 6, 7, 8, 9, 10, or 15, amino acids and an upper bound of 20, 25, 30, 35, 40, 45, or 50 amino acids). In some embodiments, one or both linkers include 32 amino acids. In some embodiments, one or both linkers includes glycine-serine linkers flanking an XTEN sequence or a sequence otherwise known to those of skill in the art.

[0277] In some embodiments, an editing system includes a deaminase associated with a DNA binding domain such as a catalytically impaired nuclease domain. In some embodiments, the DNA binding domain can localize the deaminase to a target nucleic acid in which one or more nucleotides are deaminated by the deaminase. Catalytically impaired nuclease domains are polypeptide domains that have amino acid sequences engineered from reference nuclease domain sequences but that have a reduced ability to cause double-strand breaks (DSBs) as compared to the reference (e.g., a wild type and / or fully functional nuclease) or have no ability to cause double-strand breaks. As referred to herein, a nickase refers to a catalytically impaired nuclease domain that, upon contact with a double-stranded nucleic acid substrate, cleaves one strand (e.g., a target strand) of the double-stranded nucleic acid but not both strands of the double-stranded nucleic acid. In some embodiments, a nickase, upon contact with a double-stranded nucleic acid substrate, cleaves one strand of the double-stranded nucleic acid but not both strands of the double-stranded nucleic acid in at least 70% of contacted double-stranded nucleic acid substrates (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of double- stranded nucleic acid substrates).

[0278] Base editing systems are exemplary of editing systems that include deaminase enzymes. A base editing enzyme includes a deaminase enzyme fused to a DNA binding domain that is a catalytically impaired nuclease domain (e.g., a nickase, e.g., a nickase that nicks a single strand, e.g., a non-edited strand). DNA binding domains of base editing enzymes can be RNA guided DNA binding domains, in that an RNA guide can direct the DNA binding domain to a target nucleic acid sequence. Catalytically impaired nuclease domains of a base editing enzyme can bind nucleic acids and can localize the deaminase enzyme to a target nucleic acid.

[0279] Any nuclease of a CRISPR system can be engineered to produce a catalytically impaired nuclease domain (e.g., a nickase) and used within a base editing enzyme or system. Exemplary Cas nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12; including, e.g., spCas9, dCas9, nCas9, and Cas9-SpRY), Cas9d, Cas10, Cas12 (e.g., Cas12a (e.g., LbCas12a, AsCas12a, FnCas12a, MB3Cas12a, Cas12a-M11, Cas12a-M13 (e.g., Cas12a-M13-1), Cas12a- M26 (e.g., Cas12a-M26-1), Cas12a-M28 (e.g., Cas12a-M28-1), Cas12a-M29 (e.g., Cas12a-M29-1), Cas12a-M30 (e.g., Cas12a-M30-1), Cas12a-M31 (e.g., Cas12a-M31-1), Cas12a-M32 (e.g., Cas12a-M32-1), Cas12a-M57, Cas12a-M58, Cas12a-M59, Cas12a-M60 (e.g., Cas12a-M60-9), Cas12a-M61, or Cas12a- M62), dCas12a, Cas12b, Cas12c, Cas12g, Cas12h, or Cas12i), Cas13a, Cas13b, Cas13d, Cas14, Cas-Phi, CasX, C2c3, C2c2, C2c1, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and variants thereof. Numerous forms and variants of Cas nucleases are known in the art (e.g., spCas9, dCas9, nCas9, Cas9-SpRY, Cas12a, and dCas12a) and can have distinct characteristics, including for example recognition of distinct PAMs and PAM positions.

[0280] In some embodiments, a catalytically impaired nuclease domain generates a single-stranded nick in the non-deaminated DNA strand, inducing cells to repair the non-deaminated strand using the deaminated strand as a template. To provide one example, nCas9 can create a nick in target DNA by cutting a single strand, reducing the likelihood of detrimental indel formation as compared to methods that require a double-strand break.

[0281] Particular embodiments utilize a nuclease-inactive Cas9 (dCas9) as the catalytically disabled nuclease. However, any nuclease of a CRISPR system (many of which are described above) can be disabled and used within a base editing system (e.g., dCas12a). In some embodiments, a Cas9 domain with high fidelity is selected where the Cas9 domain displays decreased electrostatic interactions between the Cas9 domain and a sugar-phosphate backbone of a DNA, as compared to a wild-type Cas9 domain. In some embodiments, a Cas9 domain (e.g., a wild type Cas9 domain) includes one or more mutations that decrease the association between the Cas9 domain and a sugar-phosphate backbone of a DNA. Cas9 domains with high fidelity are known to those skilled in the art. For example, Cas9 domains with high fidelity have been described in Kleinstiver (2016 Nature 529: 490-495) and Slaymaker (2015 Science 351: 84-88). In some embodiments, a dCas12a is used instead of a dCas9. In some embodiments, a nuclease-inactive Cas protein (e.g., a nuclease-inactive Cas12a protein lacks DNA nuclease activity but retains RNA nuclease activity).

[0282] Other DNA binding nucleases can also be used in a base editing enzyme. For example, base- editing systems can utilize zinc finger nucleases (ZFNs) (see, e.g., Urnov 2010 Nat Rev Genet.11(9): 636- 46) and transcription activator like effector nucleases (TALENs) (see, e.g., Joung 2013 Nat Rev Mol Cell Biol. 14(1): 49-55). For additional information regarding DNA-binding nucleases, see, e.g., US 2018 / 0312825.

[0283] In some embodiments, a base editing enzyme includes a DNA glycosylase inhibitor. A DNA glycosylase inhibitor can override natural DNA repair mechanisms that might otherwise repair the intended base editing. A DNA glycosylase inhibitor can be a uracil DNA glycosylase inhibitor protein (UGI). One exemplary UGI is described in Wang (1991 Gene 99:31–37). In some embodiments, a base editing enzyme can include one or more DNA glycosylase inhibitor domains (e.g., UGI domains). In some embodiments, base editing enzymes that include more than one DNA glycosylase inhibitor domain (e.g., UGI domain)can generate fewer indels and / or deaminate target nucleic acids more efficiently than base editing enzymes that includes one DNA glycosylase inhibitor domain (e.g., UGI domain) and / or no DNA glycosylase inhibitor domains (e.g., UGI domains). For example, in some embodiments, dCas9 or a Cas9 nickase can be fused to a cytidine deaminase domain and the dCas9 or Cas9 nickase can be fused to one or more UGI domains.

[0284] In some embodiments, a deaminase domain is associated with the N-terminus of a catalytically disabled nuclease. In some embodiments, a deaminase domain is associated with the N-terminus of a catalytically disabled nuclease. In some embodiments, one or more glycosylase inhibitors (e.g., UGI domain) can be associated with the C-terminus of a catalytically disabled nuclease.

[0285] Components of base editors can be fused directly (e.g., by direct covalent bond) or via linkers. For example, a catalytically disabled nuclease can be fused via a linker to the deaminase enzyme and / or a glycosylase inhibitor. Multiple glycosylase inhibitors can also be fused via linkers. As will be understood by one of ordinary skill in the art, linkers can be used to link any peptides or portions thereof.

[0286] Exemplary linkers include polymeric linkers (e.g., polyethylene, polyethylene glycol, polyamide, polyester); amino acid linkers; carbon-nitrogen bond amide linkers; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linkers; monomeric, dimeric, or polymeric aminoalkanoic acid linkers; aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, β-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid) linkers; monomeric, dimeric, or polymeric aminohexanoic acid (Ahx) linkers; carbocyclic moiety (e.g., cyclopentane, cyclohexane) linkers; aryl or heteroaryl moiety linkers; and phenyl ring linkers.

[0287] Linkers can also include functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from a peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

[0288] In some embodiments, linkers range from about 4-100 amino acids in length. In some embodiments, linkers are 4 amino acids, 9 amino acids, 14 amino acids, 16 amino acids, 32 amino acids, or 100 amino acids.

[0289] Various base editing enzymes are known in the art. Examples of base editing enzymes include BE1 (APOBEC1-16 amino acid (aa) linker-Sp dCas9 (D10A, H840A) (see, e.g., Komor 2016 Nature 533: 420–424)), BE2 (APOBEC1-16aa linker-Sp dCas9 (D10A, H840A)-4aa linker-UGI (see, e.g., Komor 2016 Nature 533: 420–424)), BE3 (APOBEC1-16aa linker-SpnCas9 (D10A)-4aa linker-UGI (see, e.g., Komor 2016 Nature 533: 420–424)), HF-BE2 (rAPOBEC1-HF2 nCas9-UGI), HF-BE3 (APOBEC1-16aa linker- HF nCas9 (D10A)-4aa linker-UGI (see, e.g., Rees 2017 Nat. Commun.8: 15790)), BE4 (rAPOBEC1-Sp nCas9-UGI-UGI), BE4max (APOBEC1-32aa linker-Sp nCas9 (D10A)-9aa linker-UGI-9aa linker-UGI(see, e.g., Koblan 2018 Nat. Biotechnol 36(9): 843-846 and / or Komor 2017 Sci. Adv. 3(8): eaao4774)), BE4-GAM (Gam-16aa linker-APOBEC1-32aa linker-Sp nCas9 (D10A)-9aa linker-UGI-9aa linker-UGI (see, e.g., Komor 2017 Sci. Adv.3(8): eaao4774)), YE1-BE3 (APOBEC1 (W90Y, R126E)-16aa linker-Sp nCas9 (D10A)-4aa linker-UGI (see, e.g., Kim 2017 Nat. Biotechnol.35: 475–480)), EE-BE3 (APOBEC1 (R126E, R132E)-16aa linker-Sp nCas9 (D10A)-4aa linker-UGI (see, e.g., Kim 2017 Nat. Biotechnol.35: 475–480)), YE2-BE3 (APOBEC1 (W90Y, R132E)-16aa linker-Sp nCas9 (D10A)-4aa linker-UGI (see, e.g., Kim 2017 Nat. Biotechnol. 35: 475–480)), YEE-BE3 (APOBEC1 (W90Y, R126E, R132E)-16aa linker-Sp nCas9 (D10A)-4aa linker-UGI (see, e.g., Kim 2017 Nat. Biotechnol.35: 475–480)), VQR-BE3 (APOBEC1-16aa linker-Sp VQR nCas9 (D10A)-4aa linker-UGI (see, e.g., Kim 2017 Nat. Biotechnol.35: 475–480)), EQR-BE3 (rAPOBEC1-EQR SpnCas9-UGI), VRER-BE3 (APOBEC1-16aa linker-Sp VRER nCas9 (D10A)-4aa linker-UGI (see, e.g., Kim 2017 Nat. Biotechnol.35: 475–480)), Sa-BE3 (APOBEC1- 16aa linker-Sa nCas9 (D10A)-4aa linker-UGI (see, e.g., Kim 2017 Nat. Biotechnol. 35: 475–480)), SA- BE4 (APOBEC1-32aa linker-Sa nCas9 (D10A)-9aa linker-UGI-9aa linker-UGI (see, e.g., Komor 2017 Sci. Adv. 3(8): eaao4774)), SaBE4-Gam (Gam-16aa linker-APOBEC1-32aa linker-Sa nCas9 (D10A)-9aa linker-UGI-9aa linker-UGI (see, e.g., Komor 2017 Sci. Adv.3(8): eaao4774)), SaKKH-BE3 (APOBEC1- 16aa linker-Sa KKH nCas9 (D10A)-4aa linker-UGI (see, e.g., Kim 2017 Nat. Biotechnol.35: 475–480)), FNLS-BE3 (rAPOBEC1-Sp nCas9-UGI), RA-BE3 (rAPOBEC1 (RA)-Sp nCas9-UGI), Cas12a-BE (APOBEC1-16aa linker-dCas12a-14aa linker-UGI (see, e.g., Li 2018 Nat. Biotechnol. 36: 324–327)), Target-AID (Sp nCas9 (D10A)-100aa linker-CDA1-9aa linker-UGI (see, e.g., Nishida 2016 Science 353(6305): aaf8729)), Target-AID-NG (Sp nCas9 (D10A)-NG-100aa linker-CDA1-9aa linker-UGI (see, e.g., Nishimasu 2018 Science 361(6408): 1259–1262)), xBE3 (APOBEC1-16aa linker-xCas9(D10A)-4aa linker-UGI (see, e.g., Hu 2018 Nature 556: 57–63)), eA3A-BE3 (APOBEC3A (N37G)-16aa linker-Sp nCas9(D10A)-4aa linker-UGI (see, e.g., Gehrke 2018 Nat. Biotechnol. 36(10): 977-982)), A3A-BE3 (hAPOBEC3A-16aa linker-Sp nCas9(D10A)-4aa linker-UGI (see, e.g., Wang 2018 Nat. Biotechnol. 36: 946–949)), eA3A-HF1-BE3-2xUGI (APOBEC3A-HF1 Sp nCas9-UGI-UGI), eA3A-HypaBE3-2xUGI (APOBEC3A-Hypa Sp nCas9-UGI-UGI), hA3A-BE3 (hAPOBEC3A-Sp nCas9-UGI), hA3B-BE3 (hAPOBEC3B-Sp nCas9-UGI), hA3G-BE3 (hAPOBEC3G-Sp nCas9-UGI), hAID-BE3 (hAPOBEC3A- Sp nCas9-UGI), SaCas9-BE3 (rAPOBEC1-SanCas9-UGI), xCas9-BE3 (rAPOBEC1-xnCas9-UGI), ScCas9-BE3 (rAPOBEC1-ScnCas9-UGI), SniperCas9-BE3 (rAPOBEC1-SnipernCas9-UGI), iSpyMac- BE3 (rAPOBEC1-iSpyMacnCas9-UGI), CRISPR-X (Sp dCas9-MS2-hAID), TAM (Sp dCas9-hAID (P182X)), AncBE4-Max (rAPOBEC1-Sp nCas9- UGI-UGI), ABE7.8 / 9 / 10 (ecTadA-ecTadA*-Sp nCas9), xCas9-ABE7.10 (ecTadA-ecTadA*-nxCas9), VQR-ABE (ecTadA-ecTadA*-Sp VQR nCas9), Sa(KKH)- ABE ecTadA-ecTadA*-Sa KKH nCas9), ABEmax (ecTadA-ecTadA*-Sp nCas9), ABE7.10max (ecTadA- ecTadA*-SpnCas9), ABE8e)ecTadA-ecTadA*-SpnCas9), PE1 (dSpCas9-MMLV-RT), PE2 (dSpCas9-MMLV-RT), PE3 (nSpCas9-MMLV-RT), and BE-PLUS (10X GCN4-Sp nCas9(D10A) / ScFv- rAPOBEC1-UGI (see, e.g., Jiang 2018 Cell Res. 28(8): 855-861)). For additional examples of BE complexes, including adenine deaminase base editors, see, e.g., Rees 2018 Nat. Rev Genet.19(12): 770- 788 and / or Kantor 2020 Int. J. Mol. Sci.21(17): 6240.

[0290] Various base editors are “dual base editors” that can edit both adenine and cytosine. Dual base editor enzymes can be fusion polypeptides that include a cytosine deaminase domain and an adenine deaminase domain. For instance, a dual base editor known as Target-ACEmax includes a codon-optimized fusion of the cytosine deaminase PmCDA1, the adenosine deaminase TadA, and a Cas9 nickase (Target- ACEmax) (see, e.g., Sakata 2020 Nature Biotechnology, 38(7), 865–869). Other exemplary dual base editors include SPACE (synchronous programmable adenine and cytosine editor). The SPACE editing enzyme is a fusion polypeptide that includes both miniABEmax-V82G and Target-AID editing domains together with a Cas9 (SpCas9-D10A) nickase domain (see, e.g., Grünewald 2020 Nat. Biotechnol.38:861– 864). A dual base editor known as A&C-BEmax includes a fusion of both cytidine and adenosine deaminase domains with a Cas9 nickase domain (see, e.g., Zhang 2020 Nat. Biotechnol.38:856–860).

[0291] For some CRISPR / Cas base editing systems, such as Cas9 or Cas14-derived systems, those systems can include a guide RNA (gRNA) that includes at least a fragment that base pairs with a complementary target nucleic acid (e.g., at least 80% identity between the fragment and the complement of the target nucleic acid, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity), where the fragment can be 10 to 40 nucleotides in length (e.g., equal to or about 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides in length, e.g., 17-24 or 17-20 nucleotides in length), e.g., where the target sequence is upstream of an appropriate PAM site. In some embodiments, a fragment of a gRNA that is complementary to a target nucleic acid sequence is positioned at the 5′ end of a gRNA or is 5′ relative to one or more other fragments of the gRNA. In some embodiments, a gRNA includes a sequence that forms a stemloop structure and binds with and / or recruits the base editor, which editor can optionally have a catalytically impaired DNA nuclease domain. A gRNA that includes both a fragment that base pairs with a complementary target nucleic acid sequence and a fragment that forms a stemloop structure and binds with and / or recruits the catalytically impaired nuclease domain of a base editing enzyme can be referred to as a single guide RNA (sgRNA). Fragments of sgRNA can be associated via a linker fragment.

[0292] A guide RNA (e.g., an sgRNA) is thought to randomly interrogate nucleic acids until it encounters a nucleic acid that is sufficiently complementary to the 5′ fragment. Upon binding of a gRNA to a DNA nucleic acid target present in double-stranded DNA, base pairing between the gRNA and target nucleic acid strand causes displacement of a small segment of single-stranded DNA. In some embodiments, the gRNA recruits the catalytically impaired nuclease domain. Nucleotides of the displaced single-strandedDNA can be modified by the deaminase enzyme. The resultant base pair can then be repaired by cellular mismatch repair machinery to a new base pair, or alternatively in some instances reverted by base excision repair mediated by uracil glycosylase. In some embodiments, a glycosylase inhibitor (e.g., UGI) reduces the occurrence of reversion.

[0293] For other CRISPR / Cas base editing systems, such as Cas12 or Cas13-derived systems, those systems can include a crRNA that includes at least a fragment that base pairs with a complementary target nucleic acid (e.g., at least 80% identity between the fragment and the complement of the target nucleic acid, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity), where the fragment can be 10 to 40 nucleotides in length (e.g., equal to or about 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides in length, e.g., 17-24 or 17-20 nucleotides in length), e.g., where the target sequence is relative to an appropriate PAM site. In some embodiments, a crRNA includes a sequence that forms a stemloop structure and binds with and / or recruits a base editor, which base editor can optionally have a catalytically impaired DNA nuclease domain.

[0294] The present disclosure includes base editing enzymes and systems engineered to increase the editing window of base editing. For example, the present disclosure includes circularly permuted base editors, described for example in Huang 2020 Nature Biotechnology, 37(6), 626–631, which is incorporated herein with respect to base editing enzymes, base editing systems, and editing windows thereof. Circularly permuted base editing enzymes and systems can be characterized by an increased range of target bases that can be modified within the protospacer up to and including, for example, at least 5, 6, 7, 8, or 9 nucleotides. For example, certain base editing systems including Cas9 variants, including cytosine and four adenine base editing enzymes, can deaminated nucleotides in a window expanded from about 4-5 nucleotides to up about 8-9 nucleotides, optionally with reduced byproduct formation.

[0295] Base editing enzymes and systems can also target and / or modify RNA molecules. One advantage of using RNA editing systems is that there is no permanent change in the genome. RNA base editors achieve analogous changes using components that base modify RNA. For example, adenosine deaminase can modify transcribed mRNA, replacing adenosine with inosine at a target site. In mammals, the most prevalent post-transcription RNA editing case is catalyzed by the adenosine deaminase enzymes (ADARs). ADAR proteins are a highly conserved family of proteins that include a single deaminase domain (DD) and one or more double-stranded RNA (dsRNA)-binding domains ADARs (e.g., ADAR1 or ADAR2) bind to dsRNA and catalyzes adenosine to inosine (A-to-I), which is read as guanosine by cellular translational machinery. ADAR1 and ADAR2 domains have been demonstrated to achieve RNA editing, e.g., in HSCs (see, e.g., Harter 2009 Nat. Immunol. 10(1): 109-115). A number of catalytically inactive Cas proteins have also been used to target RNA molecules, including Cas9, Cas9d, Cas12a, Cas13a, Cas13b, Cas13d, and Cas14.

[0296] REPAIR (RNA editing for programmable adenosine to inosine replacement) is an RNA base editing system that includes catalytically inactive Cas13 protein and the deaminase activity of ADAR2. Cas13 generally includes two HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domains, which contribute to RNA-targeted nucleolytic activity. Mutations of HEPNs abolish RNA cleavage activity while maintaining RNA targeting activity, which has been used to create an RNA base editing enzyme (e.g., REPAIR) (see, e.g., Cox 2017 Science 358:1019–1027). dCas13-ADAR2DD includes catalytically inactive dCas13 variant with RNA deaminase ADAR2 (E488Q) and can execute RNA editing for programmable A-to-I (G) replacement. RNA Editing for Specific C-to-U Exchange (RESCUE) was later developed (see, e.g., Abudayyeh 2019 Science 365:382–386). gRNAs for mRNA editing can include, e.g., a fragment complementary to a target RNA and an ADAR-recruiting fragment, such that site-directed RNA editing is achieved by recruiting ADAR to a complementary target nucleic acid. RNA-guided RNA- targeting CRISPR nuclease C2C2 (later named as Cas13a) from Leptotrichia shahii was illustrated (Abudayyeh 2016 Science 353: aaf5573).

[0297] Other examples of RNA editing systems that include ADARs can include removing the endogenous RNA-targeting domains (dsRBMS) from human adenosine deaminase and replacing them with an antisense RNA oligonucleotide to produce a recombinant enzyme that can be directed to edit a selected RNA target. In some embodiments, an ADAR2 deaminase domain is fused with an RNA-binding protein, and the sequence bound by the RNA-binding protein is associated with an antisense RNA guide oligonucleotide. In some embodiments, the RNA-binding protein is derived from λ-phage N protein-boxB RNA interaction, which normally regulates antitermination during transcription of λ-phage mRNAs. λN peptide mediates binding of the N protein, is only 22 amino acids long, and the boxB RNA hairpin that it recognizes is only 17 nucleotides long and they can bind with nanomolar affinity. Thus, in some embodiments, λN peptide can be fused to the deaminase domain of human ADAR2 (λN–DD). In some embodiments, a mutant ADAR2DD(E488Q) can be used as the deaminase domain. In some embodiments, an editing enzyme can include an ADAR deaminase domain and 2 or more λN domains (e.g., 2, 3, 4, 5, or 6 λN domains). Examples of such editing enzymes and systems are described, e.g., in Montiel-Gonzalez 2013 PNAS 110(45): 18285-18290 and Montiel-Gonzalez 2016 Nuc. Acids. Res. 44(2): e157, each of which is incorporated herein by reference with respect to editing systems.

[0298] Other examples of editing systems that include ADARs can include leveraging endogenous ADAR for programmable editing of RNA (LEAPER) editing system that employs short engineered ADAR- recruiting RNAs (arRNAs) to recruit native ADAR1 or ADAR2 deaminase enzymes to change a specific adenosine to inosine. For example, in certain some embodiments, an ADAR protein or its catalytic domain can be fused with a λN peptide. In some embodiments, an ADAR protein or its catalytic domain can be fused with a λN peptide and a SNAP-tag or a Cas protein (e.g., dCas13b). A gRNA can recruit the editingenzyme to the specific site. Further description of LEAPER editing systems can be found in Qu 2019 Nat. Biotech.1059-1069, which is incorporated herein by reference.

[0299] Base editing systems can cause point mutations without producing double-strand breaks. Base editing systems can cause point mutations without producing undesired insertions and deletions (indels). For example, a base editing system can cause indels in less than 10%, 9%, 8%, 7%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, or 0.1% of edited cells or editing events.

[0300] Those of skill in the art will appreciate that a base editing gRNA (e.g., crRNA or sgRNA including a crRNA and tracrRNA) or other targeting elements to generate a selected nucleic acid sequence modification in a target nucleic acid can be readily designed and implemented, e.g., based on available sequence information.

[0301] Base editing systems do not require double-stranded DNA breaks. Base editing systems do not require a donor fragment or template. Base editing systems provide precise control of the site at which the editing system modifies a target nucleic acid. Base editing systems can be multiplexed to achieve editing of multiple targets using a single editing enzyme, optionally including therapeutic targets. The present disclosure includes base editing systems that include a plurality of crRNAs or sgRNAs (e.g., two or more, e.g., two, three, four, or five) crRNAs or sgRNAs.

[0302] The present disclosure includes, among other things, prime editing agents and systems, and therapeutic sequences encoding the same, e.g., where the therapeutic sequence is present in an adenoviral vector or genome. A prime editing system can include a prime editing enzyme and / or at least one pegRNA as components thereof. Prime editing can introduce all possible types of point mutations, small insertions, and small deletions in a precise and targeted manner. A prime editing enzyme includes a reverse transcriptase fused to a DNA binding domain that is a catalytically impaired nuclease domain (e.g., a nickase, e.g., a nickase that nicks a single strand, e.g., a non-edited strand). A reverse transcriptase is an enzyme that can synthesize a DNA molecule from an RNA template. A reverse transcriptase generally produces a DNA molecule that is complementary to the RNA template.

[0303] In some embodiments, an editing enzyme includes an AMV reverse transcriptase, MLV reverse transcriptase, HIV-1 reverse transcriptase, or bacterial reverse transcriptase. Certain embodiments utilize an MLV reverse transcriptase domain. Reverse transcriptases of the present disclosure can have wild type amino acid sequences or engineered amino acid sequences.

[0304] Examples of reverse transcriptase enzymes include AMV reverse transcriptases (e.g., wild type AMV reverse transcriptase (RNase H plus activity), eAMVTM (engineered; RNase Hplus activity) or THermoScriptTM (engineered; reduce RNAase H activity)), MLV reverse transcriptases (e.g., wild type M-MLV reverse transcriptase, GoScriptTM, or MultiScribeTM (RNase H plus activity), AccuScript Hi-Fi (engineered, RNase H minus (3′–5′ exonuclease activity), Affinity Script (engineered;E69K / E302R / W313F / L435G / N454K; unspecified RNase H activity), ArrayScript™ (engineered; unspecified RNase H activity), BioScript™ (engineered; reduced RNase H activity), CycleScript™ (engineered), EnzScript™ (engineered; RNase H minus), EpiScript™ (engineered; RNase H minus), Expand™ reverse transcriptase (engineered; RNase H reduced), FIREScript (engineered; RNase H plus), GrandScript (engineered; RNase H plus), iScript™ (engineered; RNase H plus), Maxima™ RT (engineered; RNase H plus and minus), MonsterScript™ (engineered; RNase H minus), PrimeScript™ (engineered; RNase H minus), PrimeScript™ II (engineered; RNase H minus), PrimeScript™ III (engineered; RNase H minus), PrimeScript™ IV (engineered; RNase H minus), ProtoScript® (Engineered; RNase H plus), ProtoScript® II (engineered; RNase H reduced), qScript (engineered; RNase H plus), RevertAid™ (engineered; RNase H plus and minus), ReverTra Ace® (engineered; RNase H minus), RevertUp II™ (engineered; RNase H minus), Rocketscript™ (engineered; RNase H plus and minus), Script (engineered; RNase H minus), SMART® (engineered), SMARTScribe™ (engineered; unspecified RNase H activity), SuperScript™ II (engineered; 524G / D583N / E562Q; RNase H reduced), SuperScript™ III (engineered; 204R / V223H / T306K / F309N / D524G / D583N / E562Q; RNase H reduced), SuperScript™ IV (engineered; RNase H reduced), or Transcriptor reverse transcriptase (engineered; RNase H plus)), an HIV- 1 reverse transcriptase (e.g., HIV-1 RT (wild type of group M subtype B; RNase H plus), Biotools high retrotranscriptase (engineered group O variant (K65R / V75I); RNase H plus), or Sunscript® (engineered group O variants with changes K358R / A359G / S360A; RNase H plus and minus)), a bacterial group II intron reverse transcriptase (e.g., Marathon RT (wild type (Eubacterium rectale); lacks RNase H domain) or TGIRT®-III RT (wild type (Geobacillus stearothermophilus); lacks RNase H domain), a bacterial DNA polymerase (e.g., BcaBEST polymerase (engineered (Bacillus caldotenax DNA polymerase without 5′–3′ and 3′–5′ exonuclease activity); lacks RNase H domain), Bst 3.0 DNA polymerase (G. stearothermophilus DNA polymerase I, large fragment; lacks 5′–3′ and 3′–5′ exonuclease activity; lacks RNase H domain), RapiDxFire™ reverse transcriptase (lacks RNase H domain), Volcano2G DNA polymerase (engineered Thermus aquaticus DNA polymerase; lacks RNase H domain), or Volcano3G DNA polymerase (engineered T. aquaticus DNA polymerase; lacks RNase H domain)), SOLIScript (engineered; RNase H reduced), Omniscript® (heterodimeric RT; RNase H plus), and SensiScript® (heterodimeric RT; RNase H plus).

[0305] In some embodiments, a reverse transcriptase is a retrovirus reverse transcriptase. In some embodiments, a reverse transcriptase is a murine leukemia virus (MLV) reverse transcriptase (RT) (e.g., an engineered MLV RT). In some embodiments, a reverse transcriptase is a bacterial group II intron RT.

[0306] In some embodiments, a prime editing enzyme or system includes a reverse transcriptase associated with a DNA binding domain such as a catalytically impaired nuclease domain. In someembodiments, the DNA binding domain can localize the reverse transcriptase to a target nucleic acid in which one or more nucleotides are substituted, inserted, and / or deleted.

[0307] DNA binding domains of prime editing enzymes can be RNA guided DNA binding domains, in that an RNA guide can direct the DNA binding domain to a target nucleic acid sequence. Catalytically impaired nuclease domains of a prime editing enzyme can bind nucleic acids and can localize the reverse transcriptase enzyme to a target nucleic acid in which one or more nucleotides are substituted, inserted, and / or deleted by the prime editing system.

[0308] Any nuclease of the CRISPR system can be engineered to produce a catalytically impaired nuclease domain (e.g., a nickase) and used within a prime editing enzyme or system. Exemplary Cas nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12; including, e.g., spCas9, dCas9, nCas9, and Cas9-SpRY), Cas9d, Cas10, Cas12 (e.g., Cas12a (e.g., LbCas12a, AsCas12a, FnCas12a, MB3Cas12a, Cas12a-M11, Cas12a-M13 (e.g., Cas12a-M13-1), Cas12a- M26 (e.g., Cas12a-M26-1), Cas12a-M28 (e.g., Cas12a-M28-1), Cas12a-M29 (e.g., Cas12a-M29-1), Cas12a-M30 (e.g., Cas12a-M30-1), Cas12a-M31 (e.g., Cas12a-M31-1), Cas12a-M32 (e.g., Cas12a-M32- 1), Cas12a-M57, Cas12a-M58, Cas12a-M59, Cas12a-M60 (e.g., Cas12a-M60-9), Cas12a-M61, or Cas12a- M62), dCas12a, Cas12b, Cas12c, Cas12g, Cas12h, or Cas12i), Cas13a, Cas13b, Cas13d, Cas14, Cas-Phi, CasX, C2c3, C2c2, C2c1, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and variants thereof. Numerous forms and variants of Cas nucleases are known in the art (e.g., spCas9, dCas9, nCas9, Cas9-SpRY, Cas12a, and dCas12a) and can have distinct characteristics, including for example recognition of distinct PAMs and PAM positions.

[0309] Other DNA binding nucleases can also be used in a prime editing enzyme. For example, prime editing systems can utilize zinc finger nucleases (ZFNs) (see, e.g., Urnov 2010 Nat Rev Genet.11(9): 636- 46) and transcription activator like effector nucleases (TALENs) (see, e.g., Joung 2013 Nat Rev Mol Cell Biol. 14(1): 49-55). For additional information regarding DNA-binding nucleases, see, e.g., US 2018 / 0312825.

[0310] In some embodiments, a prime editing system includes a prime editing gRNA (pegRNA) that specifies a target nucleic acid sequence and also specifies the sequence modification that the prime editing system introduces. The pegRNA includes a sequence complimentary to the target nucleic acid and recruits the prime editing enzyme to the target nucleic acid. A pegRNA includes, from 5′ to 3′: (a) a fragment that base pairs with a complementary target nucleic acid sequence (e.g., at least 80% identity between the fragment and the complement of the target nucleic acid, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) (sometimes referred to as a “spacer”), where the fragment can be 10 to 40 nucleotides in length (e.g., equal to or about 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35or 40 nucleotides in length, e.g., 17-24 or 17-20 nucleotides in length); (b) a sequence that forms a stemloop structure and binds with and / or recruits the catalytically impaired nuclease domain of a prime editing enzyme; (c) a fragment that includes a sequence that includes one or more modifications (e.g., one or more substitutions, insertions, and / or deletions) relative to the target nucleic acid sequence (sometimes referred to as a “template sequence”), and is complementary (excepting modifications) to the same target nucleic acid strand as (d); and (d) a fragment that includes a sequence complimentary to a target sequence (sometimes referred to as a “binding region” or “primer binding site” (PBS)), e.g., where the target sequence is upstream of an appropriate PAM site. In some embodiments, a PBS can be 5 to 20 nucleotides, e.g., 8 to 15 nucleotides in length. In some embodiments, a template sequence can be 10 to 20 nucleotides in length, or longer. Because pegRNAs include components characteristic of sgRNAs, they are sometimes described as extended sgRNAs. Any two fragments of a pegRNA can be, independently, associated directly or via a linker fragment.

[0311] A catalytically impaired nuclease domain of a prime editing enzyme can nick a target nucleic acid that includes an appropriate PAM to expose a 3′ flap and a 5′ flap. After nicking of the target nucleic acid, the released 3′ flap can hybridize to the PBS of the pegRNA, priming reverse transcription of the template fragment of the pegRNA that includes a modification of the target sequence, directly introducing the modification into the target nucleic acid to the 3′ flap. The product of reverse transcription, an edited 3′ flap that is “redundant” with the 5′ flap sequence produced by the nick (which includes the original, unedited sequence of the target nucleic acid), can then compete with the original and redundant 5′ flap sequence for reincorporation into the DNA duplex. Although the perfectly complimentary 5′ would likely be thermodynamically favored for hybridization to the non-edited strand, the 5′ flap is preferentially degraded by cellular endonucleases that are ubiquitous during lagging-strand DNA synthesis. After 5′ flap excision and ligation of the edited strand, permanent installation of the edit occurs through DNA repair of the non-edited that relies on the edited strand as a template. DNA repair of the non-edited strand can be promoted by contact with a secondary sgRNA that directs nicking of the non-edited strand. This additional nick stimulates re-synthesis of the non-edited strand using the edited strand as a template, resulting in a fully edited duplex. Prime editing systems can introduce any of one or more of the 12 types of point mutations (all possible nucleotide transitions and transversions), as well as insertions and / or deletions.

[0312] In some embodiments, a prime editing system is engineered to disrupt a PAM site of a target nucleic acid. Disruption of a PAM site of a target nucleic acid can reduce the probability of repeated editing of the particular target nucleic acid. In some embodiments, disruption of a PAM site in edited target nucleic acids can increase the efficiency of prime editing and / or gene therapy that includes prime editing.

[0313] Exemplary prime editing systems include PE1, PE2, and PE3. Each of these prime editing enzymes include a mutant Streptococcus pyogenes Cas9 nickase domain (H840A mutant) and a Moloneymurine leukemia virus (M-MLV) reverse transcriptase (e.g., engineered to include D200N / T306K / W313F / T330P / L603W). PE1 includes a pegRNA and a prime editing enzyme that includes a Cas9 H840A nickase and wild type MLV RT. The Cas9 nickase acts only on the strand to be edited by the RT. PE2 includes pegRNA and a prime editing enzyme that includes a Cas9 H840A nickase and engineered MLV RT (D200N / T306K / W313F / T330P / L603W) demonstrated to improve editing efficiency. PE3 includes the same prime editing enzyme as PE2 (as well as a pegRNA) but further includes an sgRNA that targets the non-edited strand for nicking 14-116 nucleotides away from the site of the pegRNA-induced nick (PE3), where cellular mismatch repair pathways can fix the information introduced in the edited strand. Compared with PE2, the PE3b strategy demonstrate increased editing efficiency and lower levels of indel formation. A variant of the PE3 system called PE3b uses a nicking sgRNA that targets only the edited sequence, resulting in decreased levels of indel products by preventing nicking of the non-edited DNA strand until the other strand has been converted to the edited sequence.

[0314] Those of skill in the art will appreciate that a pegRNA or other targeting elements to generate a selected nucleic acid sequence modification in a target nucleic acid can be readily designed and implemented, e.g., based on available sequence information. Various tools for designing pegRNAs are available. For example, pegFinder is a web-based tool for pegRNA design (see, e.g., Chow 2020 Nat. Biomed. Eng. doi: 10.1038 / s41551-020-00622-8). Another example of a web-based tool for pegRNA design is PrimeDesign (see, e.g., Hsu 2020 bioRxiv doi: 10.1101 / 2020.05.04.077750).

[0315] Prime editing systems do not require double-stranded DNA breaks. Prime editing systems provide precise control of the site at which the editing system modifies a target nucleic acid. Prime editing systems can be multiplexed to achieve editing of multiple targets using a single editing enzyme, optionally including therapeutic targets. The present disclosure includes that a prime editing system can include a plurality of pegRNAs (e.g., two or more, e.g., two, three, four, or five pegRNAs).

[0316] The present disclosure includes, among other things, zinc finger nucleases, and therapeutic sequences encoding the same, e.g., where the therapeutic sequence is present in an adenoviral vector or genome. Zinc finger nucleases (ZFNs) are artificial restriction enzymes made by associating a sequence- targeted zinc-finger DNA-binding units with a nuclease domain (e.g., Fok1 nuclease domain) in a fusion protein. Each ZFN includes a nuclease domain (e.g., the cleavage domain of FokI) linked to an array of three to six zinc fingers zinc fingers (ZFs). For example, a ZFN can include several Cys2His2 ZFs in which each unit includes about 30 amino acids and specifically binds about 3 nucleotides. The ZFs provide a ZFN with the ability to bind a particular nucleic acid sequence. Because the FokI cleavage domain must dimerize to cut DNA, a monomer is not active, and cleavage does not occur at single binding sites. Thus, for example, ZFNs including three ZFs that together bind a 9-bp target function as ZFN dimers that specifically bind 18 bp of DNA per cleavage site. In some embodiments, ZFNs can include up to six ZFs per ZFN.

[0317] Cleavage of a target nucleic acid by ZFNs induces cellular repair processes that can mediate modification of the nucleic acid. ZFN-induced double-strand breaks can lead to both targeted modification and targeted gene replacement. For example, if a ZFN-induced cleavage is resolved by non-homologous end joining, this can result in small deletions or insertions, which can lead to gene knockout. If a ZFN- induced cleavage is resolved by a homology-based process in the presence of a provided donor nucleic acid, small changes (e.g., one or a few nucleotides) or more (e.g., up to and including entire transgenes) can be introduced into the target nucleic acid.

[0318] The present disclosure includes, among other things, Transcription Activator-Like Effector Nuclease (TALEN) editing agents and systems, and therapeutic sequences encoding the same, e.g., where the therapeutic sequence is present in an adenoviral vector or genome. Various editing enzymes and systems can include a transcription activator-like (TAL) effector DNA binding domain and an endonuclease enzyme. An editing enzyme including a TAL effector DNA binding domain and an endonuclease can be referred to as a TALEN.

[0319] TAL effector DNA binding domains includes a plurality of monomers, each of which monomers binds one nucleotide in the target nucleic acid sequence. Each monomer includes 34 amino acids. In each monomer, positions 12 and 13 (referred to as the repeat variable diresidue, RVD) are highly variable and contribute to specific recognition of different nucleotides. The final monomer of a TAL effector DNA binding domain, which binds the nucleotide at the 3’-end of the recognition site, can be only 20 amino acids in length and therefore is sometimes referred to as a half-repeat. RVD sequences can be degenerate, as certain RVD combinations can bind to two or more nucleotides, e.g., with distinct efficiency. For example, RVDs include Asn and Ile (NI), Asn and Gly (NG), Asn and Asn (NN), and His and Asp (HD), which bind A, T, G, and C nucleotides, respectively.

[0320] In some embodiments, a TAL effector DNA binding domain is isolated from Xanthomonas spp. In some embodiments, a TALEN includes an endonuclease domain (e.g., a FokI domain), e.g., C- terminal to the TAL effector DNA binding domain.

[0321] TALENs work as pairs, the two members having target binding site on opposite DNA strands of the target nucleic acid sequence, with the targets separated by a small fragment (e.g., 12–25 bp) that can be referred to as a spacer sequence. Once a pair of TALENs have bound their target sites, the endonuclease (e.g., FokI) domains dimerize and cause a double-strand break in a spacer sequence. Non-homologous end joining (NHEJ) to resolve a DSB directly ligates DNA from either side of the double-strand break where there is very little or no sequence overlap for annealing. This repair mechanism can cause indels (insertion or deletion), or chromosomal rearrangement, which can disrupt genes at that target nucleic acid sequence. Alternatively, DNA can be introduced into a genome through NHEJ in the presence of exogenous double-stranded DNA fragments. Homology directed repair can also introduce foreign DNA at the DSB as the transfected double-stranded sequences are used as templates for the repair enzymes.

[0322] In some embodiments, a therapeutic sequence encodes a therapeutic nucleic acid. In some embodiments, a therapeutic nucleic acid comprises or is a small RNA. Small RNAs are short, non-coding RNA molecules that play a role in regulating gene expression. In some embodiments, small RNAs are less than 200 nucleotides in length. In some embodiments, small RNAs are less than 100 nucleotides in length. In some embodiments, small RNAs are less than 50, 45, 40, 35, 30, 25, or 20 nucleotides in length. In some embodiments, small RNAs are less than 20 nucleotides in length. In some embodiments, a small RNA has a length having a lower bound of 5, 10, 15, 20, 25, or 30 nucleotides and an upper bound of 20, 25, 30, 35, 40, 45, 50, 75, or 100 nucleotides. Small RNAs include but are not limited to microRNAs (miRNAs, Piwi- interacting RNAs (piRNAs), small interfering RNAs (siRNAs), small nucleolar RNAs (snoRNAs), tRNA- derived small RNAs (tsRNAs) small rDNA-derived RNAs (srRNAs), and small nuclear RNAs. Additional classes of small RNAs continue to be discovered.

[0323] In some embodiments, interfering RNA molecules that are homologous to a target mRNA or to which the interfering RNA can hybridize can lead to degradation of the target mRNA molecule or reduced translation of the target mRNA, a process referred to as RNA interference (RNAi) (Carthew, Curr. Opin. Cell. Biol. 13: 244-248, 2001). RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). In some instances, natural RNAi proceeds via fragments cleaved from free double-strand RNA (dsRNA) which direct the degradative mechanism to other similar RNA sequences. Alternatively, RNAi can be manufactured, for example, to silence the expression of target genes. Exemplary RNAi molecules include small hairpin RNA (shRNA, also referred to as short hairpin RNA) and small interfering RNA (siRNA).

[0324] Without limiting the disclosure, and without being bound by theory, RNA interference in nature and / or in some embodiments is typically a two-step process. In the first step, the initiation step, input dsRNA is digested into 21-23 nucleotide (nt) siRNA, probably by the action of Dicer, a member of the ribonuclease (RNase) III family of dsRNA-specific ribonucleases, which processes (cleaves) dsRNA (introduced directly or via a transgene or a virus) in an ATP-dependent manner. Successive cleavage events degrade the RNA to 19-21 base pair (bp) duplexes (siRNA), each with 2-nucleotide 3' overhangs.

[0325] In a second step, an effector step, the siRNA duplexes bind to a nuclease complex to form the RNA-induced silencing complex (RISC). An ATP-dependent unwinding of the siRNA duplex is required for activation of the RISC. The active RISC then targets the homologous transcript by base pairing interactions and typically cleaves the mRNA into 12 nucleotide fragments from the 3' terminus of the siRNA. Research indicates that each RISC contains a single siRNA and an RNase.

[0326] Because of the remarkable potency of RNAi, an amplification step within the RNAi pathway has been suggested. Amplification could occur by copying of the input dsRNAs which would generate more siRNAs, or by replication of the siRNAs formed. Alternatively or additionally, amplification could be affected by multiple turnover events of the RISC.

[0327] ShRNAs are single-stranded polynucleotides with a hairpin loop structure. The single-stranded polynucleotide has a loop segment linking the 3' end of one strand in the double-stranded region and the 5' end of the other strand in the double-stranded region. The double-stranded region is formed from a first sequence that is hybridizable to a target sequence, such as a polynucleotide encoding transgene, and a second sequence that is complementary to the first sequence, thus the first and second sequence form a double stranded region to which the linking sequence connects the ends of to form the hairpin loop structure. The first sequence can be hybridizable to any portion of a polynucleotide encoding transgene. The double- stranded stem domain of the shRNA can include a restriction endonuclease site.

[0328] Transcription of shRNAs is initiated at a polymerase III (Pol III) promoter and is thought to be terminated at position 2 of a 4-5-thymine transcription termination site. Upon expression, shRNAs are thought to fold into a stem-loop structure with 3′ UU-overhangs; subsequently, the ends of these shRNAs are processed, converting the shRNAs into siRNA-like molecules of 21-23 nucleotides.

[0329] The stem-loop structure of shRNAs can have optional nucleotide overhangs, such as 2-bp overhangs, for example, 3' UU overhangs. While there may be variation, stems typically range from 15 to 49, 15 to 35, 19 to 35, 21 to 31 bp, or 21 to 29 bp, and the loops can range from 4 to 30 bp, for example, 4 to 23 bp. In some embodiments, shRNA sequences include 45-65 bp; 50-60 bp; or 51, 52, 53, 54, 55, 56, 57, 58, or 59 bp. In some embodiments, shRNA sequences include 52 or 55 bp. In some embodiments, siRNAs have 15-25 bp. In some embodiments, siRNAs have 16, 17, 18, 19, 20, 21, 22, 23, or 24 bp. In some embodiments, siRNAs have 19 bp. The skilled artisan will appreciate, however, that siRNAs having a length of less than 16 nucleotides or greater than 24 nucleotides can also function to mediate RNAi. Longer RNAi agents have been demonstrated to elicit an interferon or Protein kinase R (PKR) response in certain mammalian cells which may be undesirable. Preferably the RNAi agents do not elicit a PKR response (i.e., are of a sufficiently short length). However, longer RNAi agents may be useful, for example, in situations where the PKR response has been downregulated or dampened by alternative means.

[0330] In some embodiments, a therapeutic sequence can be selected to provide a therapeutically effective response against chronic granulomatous disease (CGD). For example, in some embodiments, a therapeutic encodes a therapeutic polypeptide that comprises or is CYBB, CYBA, NCF1, NCF2, NCF4, or CYBC1.

[0331] In some embodiments, a therapeutic sequence comprises a nucleic acid sequence encoding a CYBB therapeutic polypeptide. In some embodiments, a therapeutic sequence is at least about 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 79. In some embodiments, a therapeutic sequence comprises a sequence of SEQ ID NO: 79. In some embodiments, a therapeutic sequence has a nucleic acid sequence of SEQ ID NO: 79. In some embodiments, a therapeutic sequence encoding a CYBB therapeutic polypeptide comprises a nucleic acid sequence encoding a CYBB polypeptide comprising an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 151. In some embodiments, a therapeutic sequence encoding a CYBB therapeutic polypeptide comprises a nucleic acid sequence encoding a CYBB therapeutic polypeptide comprising an amino acid sequence of SEQ ID NO: 151. In some embodiments, a therapeutic sequence encoding a CYBB therapeutic polypeptide comprises a nucleic acid sequence encoding a CYBB polypeptide that has an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 151. In some embodiments, a therapeutic sequence encoding a CYBB therapeutic polypeptide comprises a nucleic acid sequence encoding a CYBB therapeutic polypeptide that has an amino acid sequence of SEQ ID NO: 151.

[0332] In some embodiments, a therapeutic sequence comprises a nucleic acid sequence encoding a CYBA therapeutic polypeptide. In some embodiments, a therapeutic sequence encoding a CYBA therapeutic polypeptide comprises a nucleic acid sequence encoding a CYBA polypeptide comprising an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 152. In some embodiments, a therapeutic sequence encoding a CYBA therapeutic polypeptide comprises a nucleic acid sequence encoding a CYBA therapeutic polypeptide comprising an amino acid sequence of SEQ ID NO: 152. In some embodiments, a therapeutic sequence encoding a CYBA therapeutic polypeptide comprises a nucleic acid sequence encoding a CYBA polypeptide that has an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 152. In some embodiments, a therapeutic sequence encoding a CYBA therapeutic polypeptide comprises a nucleic acid sequence encoding a CYBA therapeutic polypeptide that has an amino acid sequence of SEQ ID NO: 152.

[0333] In some embodiments, a therapeutic sequence comprises a nucleic acid sequence encoding a CYBC1 therapeutic polypeptide. In some embodiments, a therapeutic sequence encoding a CYBC1 therapeutic polypeptide comprises a nucleic acid sequence encoding a CYBC1 polypeptide comprising an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 153. In some embodiments, a therapeutic sequence encoding a CYBC1 therapeutic polypeptide comprises a nucleic acid sequence encoding a CYBC1 therapeutic polypeptide comprising an amino acid sequence of SEQ ID NO: 153. In some embodiments, a therapeutic sequence encoding a CYBC1 therapeutic polypeptide comprises a nucleic acid sequence encoding a CYBC1 polypeptide that has an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or moreidentity to the amino acid sequence of SEQ ID NO: 153. In some embodiments, a therapeutic sequence encoding a CYBC1 therapeutic polypeptide comprises a nucleic acid sequence encoding a CYBC1 therapeutic polypeptide that has an amino acid sequence of SEQ ID NO: 153. In some embodiments, a therapeutic sequence encoding a CYBC1 therapeutic polypeptide comprises a nucleic acid sequence encoding a CYBC1 polypeptide comprising an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 154. In some embodiments, a therapeutic sequence encoding a CYBC1 therapeutic polypeptide comprises a nucleic acid sequence encoding a CYBC1 therapeutic polypeptide comprising an amino acid sequence of SEQ ID NO: 154. In some embodiments, a therapeutic sequence encoding a CYBC1 therapeutic polypeptide comprises a nucleic acid sequence encoding a CYBC1 polypeptide that has an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 154. In some embodiments, a therapeutic sequence encoding a CYBC1 therapeutic polypeptide comprises a nucleic acid sequence encoding a CYBC1 therapeutic polypeptide that has an amino acid sequence of SEQ ID NO: 154.

[0334] In some embodiments, a therapeutic sequence comprises a nucleic acid sequence encoding a NCF1 therapeutic polypeptide. In some embodiments, a therapeutic sequence encoding a NCF1 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF1 polypeptide comprising an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 155. In some embodiments, a therapeutic sequence encoding a NCF1 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF1 therapeutic polypeptide comprising an amino acid sequence of SEQ ID NO: 155. In some embodiments, a therapeutic sequence encoding a NCF1 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF1 polypeptide that has an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 155. In some embodiments, a therapeutic sequence encoding a NCF1 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF1 therapeutic polypeptide that has an amino acid sequence of SEQ ID NO: 155.

[0335] In some embodiments, a therapeutic sequence comprises a nucleic acid sequence encoding a NCF2 therapeutic polypeptide. In some embodiments, a therapeutic sequence encoding a NCF2 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF2 polypeptide comprising an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 156. In some embodiments, a therapeutic sequence encoding a NCF2 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF2 therapeutic polypeptide comprising an amino acid sequence of SEQ ID NO: 156. In some embodiments, a therapeutic sequence encoding a NCF2 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF2 polypeptide that has an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence ofSEQ ID NO: 156. In some embodiments, a therapeutic sequence encoding a NCF2 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF2 therapeutic polypeptide that has an amino acid sequence of SEQ ID NO: 156. In some embodiments, a therapeutic sequence encoding a NCF2 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF2 polypeptide comprising an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 157. In some embodiments, a therapeutic sequence encoding a NCF2 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF2 therapeutic polypeptide comprising an amino acid sequence of SEQ ID NO: 157. In some embodiments, a therapeutic sequence encoding a NCF2 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF2 polypeptide that has an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 157. In some embodiments, a therapeutic sequence encoding a NCF2 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF2 therapeutic polypeptide that has an amino acid sequence of SEQ ID NO: 157. In some embodiments, a therapeutic sequence encoding a NCF2 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF2 polypeptide comprising an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 158. In some embodiments, a therapeutic sequence encoding a NCF2 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF2 therapeutic polypeptide comprising an amino acid sequence of SEQ ID NO: 158. In some embodiments, a therapeutic sequence encoding a NCF2 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF2 polypeptide that has an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 158. In some embodiments, a therapeutic sequence encoding a NCF2 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF2 therapeutic polypeptide that has an amino acid sequence of SEQ ID NO: 158. In some embodiments, a therapeutic sequence encoding a NCF2 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF2 polypeptide comprising an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 159. In some embodiments, a therapeutic sequence encoding a NCF2 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF2 therapeutic polypeptide comprising an amino acid sequence of SEQ ID NO: 159. In some embodiments, a therapeutic sequence encoding a NCF2 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF2 polypeptide that has an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 159. In some embodiments, a therapeutic sequence encoding a NCF2 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF2 therapeutic polypeptide that has an amino acid sequence of SEQ ID NO: 159.

[0336] In some embodiments, a therapeutic sequence comprises a nucleic acid sequence encoding aNCF4 therapeutic polypeptide. In some embodiments, a therapeutic sequence encoding a NCF4 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF4 polypeptide comprising an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 160. In some embodiments, a therapeutic sequence encoding a NCF4 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF4 therapeutic polypeptide comprising an amino acid sequence of SEQ ID NO: 160. In some embodiments, a therapeutic sequence encoding a NCF4 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF4 polypeptide that has an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 160. In some embodiments, a therapeutic sequence encoding a NCF4 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF4 therapeutic polypeptide that has an amino acid sequence of SEQ ID NO: 160. In some embodiments, a therapeutic sequence encoding a NCF4 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF4 polypeptide comprising an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 161. In some embodiments, a therapeutic sequence encoding a NCF4 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF4 therapeutic polypeptide comprising an amino acid sequence of SEQ ID NO: 161. In some embodiments, a therapeutic sequence encoding a NCF4 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF4 polypeptide that has an amino acid sequence that has at least about 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 161. In some embodiments, a therapeutic sequence encoding a NCF4 therapeutic polypeptide comprises a nucleic acid sequence encoding a NCF4 therapeutic polypeptide that has an amino acid sequence of SEQ ID NO: 161. Table 10: Exemplary Therapeutic Sequences

[0337] A payload cassette can comprise one or more therapeutic sequences encoding one or more therapeutic polypeptides and / or therapeutic nucleic acids. To provide expression of a therapeutic sequence, a payload cassette may comprise one or more regulatory sequences, e.g., promoters, enhancers, polyadenylation signal sequences. In some embodiments, a payload cassette comprises a promoter. In some embodiments, a payload cassette comprises an enhancer. In some embodiments, a payload cassette comprises a polyadenylation signal sequence.

[0338] Various promoters can be included in a payload cassette to drive expression of a therapeutic sequence in target cells. In some embodiments, a payload cassette comprises a lineage-specific promoter. In some embodiments, a payload cassette comprises a lineage-specific promoter operably linked to aselection marker. In some embodiments, a payload cassette comprises a CTSG-FES promoter. In some embodiments, a payload cassette comprises a CTSG-FES promoter, wherein the CTSG-FES promoter is operably linked to a therapeutic sequence. In some embodiments, a CTSG-FES promoter is operably linked to a therapeutic sequence and drives expression of the therapeutic sequence in myeloid cells. In some embodiments, a CTSG-FES promoter is operably linked to a therapeutic sequence encoding a CYBB polypeptide, a CYBA polypeptide, a NCF1 polypeptide, or a NCF2 polypeptide. In some embodiments, a payload cassette comprises a CTSG-FES promoter comprising a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 88 or 89. In some embodiments, a payload cassette comprises a CTSG-FES promoter comprising a sequence of SEQ ID NO: 88 or 89. In some embodiments, a payload cassette comprises a CTSG-FES promoter that has a nucleic acid sequence of SEQ ID NO: 88 or 89.

[0339] Various regulatory elements can be included in a payload cassette provided herein. In some embodiments, a regulatory element is located downstream of a therapeutic sequence. In some embodiments, a regulatory element is located in a 3’ UTR of a payload cassette. In some embodiments, a regulatory element is capable of increasing level of expression of a therapeutic sequence in a payload cassette.

[0340] In some embodiments, a regulatory element comprises or is a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). Various WPRE sequences are known in the art and can be used in a payload cassette provided herein. In some embodiments, a payload cassette comprises a WPRE that comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 128. In some embodiments, a payload cassette comprises a WPRE that comprises a sequence of SEQ ID NO: 128. In some embodiments, a payload cassette comprises a WPRE that has a nucleic acid sequence of SEQ ID NO: 128.

[0341] Various polyadenylation (polyA) signal sequences are known in the art and can be used in a payload cassette provided herein. For example, in some embodiments, a polyA signal sequence comprises or is a bGH, hGH, hBG, or SV40 polyA signal sequence. In some embodiments, a polyA signal sequence is located downstream of a therapeutic sequence. In some embodiments, a polyA signal sequence is located in a 3’ UTR of a payload cassette.

[0342] In some embodiments, a payload cassette comprises a bovine growth hormone (bGH) polyA signal sequence. In some embodiments, a payload cassette comprises a polyA signal sequence, e.g., a bGH polyA signal sequence, that comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 129, 130, or 173. In some embodiments, a payload cassette comprises a polyA signal sequence, e.g., a bGH polyA signal sequence that comprises a sequence of SEQ ID NO: 129, 130, or 173. In some embodiments, a payload cassettecomprises a polyA signal sequence, e.g., a bGH polyA signal sequence that has a nucleic acid sequence of SEQ ID NO: 129, 130, or 173.

[0343] In some embodiments, a payload cassette comprises a human beta globin (hBG) polyA signal sequence. In some embodiments, a payload cassette comprises a polyA signal sequence, e.g., a hBG polyA signal sequence, that comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 131. In some embodiments, a payload cassette comprises a polyA signal sequence, e.g., a hBG polyA signal sequence that comprises a sequence of SEQ ID NO: 131. In some embodiments, a payload cassette comprises a polyA signal sequence, e.g., a hBG polyA signal sequence that has a nucleic acid sequence of SEQ ID NO: 131.

[0344] In some embodiments, a payload cassette comprises an SV40 polyA signal sequence. In some embodiments, a payload cassette comprises a polyA signal sequence, e.g., an SV40 polyA signal sequence, that comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 132 or 174. In some embodiments, a payload cassette comprises a polyA signal sequence, e.g., an SV40 polyA signal sequence that comprises a sequence of SEQ ID NO: 132 or 174. In some embodiments, a payload cassette comprises a polyA signal sequence, e.g., an SV40 polyA signal sequence that has a nucleic acid sequence of SEQ ID NO: 132 or 174.

[0345] In some embodiments, a payload cassette comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 80. In some embodiments, a payload cassette comprises a sequence of SEQ ID NO: 80. In some embodiments, a payload cassette has a nucleic acid sequence of SEQ ID NO: 80. Table 11: Exemplary Payload Cassette SequencesSelection cassettes and selection markers

[0346] Payload vectors can comprise one or more selection cassettes, which comprise one or more nucleic acid sequences encoding one or more selection markers for expression in target cells. In some embodiments, a payload vector comprises one or more selection cassettes. In some embodiments, a payload vector comprises a genome that comprises one or more selection cassettes.

[0347] A selection cassette is engineered in order to provide expression of a selection marker that can be utilized to enrich for modified target cells, e.g., target cells wherein the selection cassette has been integrated into the target cell genomes, by providing an advantage over unmodified target cells. In some embodiments, an advantage comprises reduced toxicity upon contacting of a target cell with a selectionagent. In some embodiments, unmodified target cells have reduced survival and / or proliferation as compared to modified target cells, e.g., target cells wherein the selection cassette has been integrated into the target cell genomes.

[0348] A selection cassette can include a nucleic acid sequence encoding one or more selection markers, e.g., polypeptides, proteins, that (a) confer resistance to antibiotics or other toxins, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media. Any number of selection markers can be used to recover, select for, and / or enrich for transduced cells.

[0349] In some embodiments, a positive selection marker includes resistance genes to neomycin, hygromycin, ampicillin, puromycin, phleomycin, zeomycin, blasticidin, or viomycin. In some embodiments, a positive selection marker includes the DHFR (dihydrofolate reductase) gene providing resistance to methotrexate, a MGMTP140Kgene responsible for the resistance to O6BG / BCNU, a HPRT (Hypoxanthine phosphoribosyl transferase) gene responsible for the transformation of specific bases present in HAT selection medium (hypoxanthine, aminopterin, thymidine), and other genes for detoxification with respect to some drugs. In some embodiments, a positive selection marker includes a modified polypeptide, e.g., a modified cell surface polypeptide, that is expressed to avoid recognition by, e.g., an antibody or antigen-binding fragment thereof, an antibody-drug conjugate (ADC).

[0350] In some embodiments, a negative selection marker includes a nucleic acid sequence encoding an expression product that transforms a substrate present in (e.g., delivered to) a subject or system (e.g., a culture medium) into a toxic substance, thereby sensitizing cells that expresses the gene. In some embodiments, for example, a payload is engineered such that proper integration into a target genome disrupts expression of a negative selection marker. A negative selection marker can include a gene encoding diphtheria toxin A-fragment (DTA) (Yagi et al., Anal Biochem.214(1): 77-86, 1993; Yanagawa et al., Transgenic Res. 8(3): 215-221, 1999) or a thymidine kinase gene of the Herpes virus (HSV TK) sensitive to the presence of ganciclovir or FIAU. In some embodiments, a negative selection cassette includes an HPRT gene for negative selection in the presence of 6-thioguanine (6TG).

[0351] In some embodiments, a selection marker comprises or is MGMTP140K. The MGMT gene encodes human alkyl guanine transferase (hAGT), a DNA repair protein that confers resistance to the cytotoxic effects of alkylating agents, such as nitrosoureas and temozolomide (TMZ). 6-benzylguanine (O6BG) is an inhibitor of AGT that potentiates nitrosourea toxicity and is co-administered with TMZ to potentiate the cytotoxic effects of this agent. Several mutant forms of MGMT that encode variants of AGT are highly resistant to inactivation by O6BG but retain their ability to repair DNA damage (Maze et al., J. Pharmacol. Exp. Ther. 290: 1467-1474, 1999). MGMTP140K-based drug-resistant gene therapy has been shown to confer chemoprotection to mouse, canine, rhesus macaques, and human cells, specifically hematopoietic cells. See, e.g., Zielske et al., J. Clin. Invest. 112: 1561-1570, 2003; Pollok et al., Hum.Gene Ther. 14: 1703-1714, 2003; Gerull et al., Hum. Gene Ther. 18: 451-456, 2007; Neff et al., Blood 105: 997-1002, 2005; Larochelle et al., J. Clin. Invest. 119: 1952-1963, 2009; Sawai et al., Mol. Ther.3: 78-87, 2001, Olszko et al. Gene Therapy 22: 591-595, 2015, which are all hereby incorporated by reference in their entirety.

[0352] In some embodiments, a nucleic acid sequence encoding a selection marker comprises a nucleic acid sequence encoding MGMTP140K. In some embodiments, a nucleic acid sequence encoding a selection marker comprises a nucleic acid sequence encoding MGMTP140Kis at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 81. In some embodiments, a nucleic acid sequence encoding a selection marker comprises a sequence of SEQ ID NO: 81. In some embodiments, a nucleic acid sequence encoding a selection marker has a nucleic acid sequence of SEQ ID NO: 81. In some embodiments, a nucleic acid sequence encoding a selection marker encodes a MGMTP140Kpolypeptide comprising an amino acid sequence of SEQ ID NO: 82. In some embodiments, a nucleic acid sequence encoding a selection marker encodes a MGMTP140Kpolypeptide that has an amino acid sequence of SEQ ID NO: 82.

[0353] A CD117 polypeptide comprising one or more mutations can be used as a selection marker in conjunction with provided technologies (e.g., vectors, genomes, selection cassettes). In some embodiments, a CD117 polypeptide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mutations). In some embodiments, a CD117 polypeptide comprises one or more mutations. In some embodiments, a CD117 polypeptide comprises two or more mutations. In some embodiments, a CD117 polypeptide comprises three or more mutations. In some embodiments, a CD117 polypeptide comprises four or more mutations. In some embodiments, a CD117 polypeptide comprises five or more mutations.

[0354] Various CD117 polypeptides, e.g., CD117 polypeptides comprising one or more mutations, that can be used in accordance with the present disclosure have been described in the art including those in WO 2021 / 041945, WO 2022 / 173861, WO 2023 / 069961, and WO 2023 / 159136, which are all hereby incorporated by reference in their entirety. In some embodiments, a CD117 polypeptide comprises or is a CD117 polypeptide described in WO 2021 / 041945. In some embodiments, a CD117 polypeptide comprises or is a CD117 polypeptide described in WO 2022 / 173861. In some embodiments, a CD117 polypeptide comprises or is a CD117 polypeptide described in WO 2023 / 069961. In some embodiments, a CD117 polypeptide comprises or is a CD117 polypeptide described in WO 2023 / 159136.

[0355] A CD117 polypeptide may comprise a mutation within an epitope targeted by an anti-CD117 antibody or antigen-binding fragment thereof. Inclusion of one or more mutations within a targeted epitope can reportedly disrupt binding of an anti-CD117 antibody or antigen-binding fragment thereof. This binding disruption can thus allow for those cells expressing a mutant CD117 polypeptide to evade ablation in a subject receiving an anti-CD117 antibody or antigen-binding fragment thereof. For example, a targetcell, e.g., a HSC or cell derived therefrom, modified using a method of in vivo gene therapy herein and expressing a mutant CD117 polypeptide as a selection marker can avoid depletion by administration an anti-CD117 antibody or antigen-binding fragment thereof. Meanwhile, unmodified target cells, e.g., unmodified HSCs or cells derived therefrom are depleted, thus providing enrichment of the modified target cells, e.g., modified HSCs or cells derived therefrom.

[0356] In some embodiments, a CD117 polypeptide comprises one or more mutations selected from F316S, M318V, I319K, V323I, I334V, E360K, P363V, E366D, E376Q, and H378R. See, e.g., Casirati, G., et al. Nature. 2023;621(7978):404-414. In some embodiments, a CD117 polypeptide comprises a F316S mutation. In some embodiments, a CD117 polypeptide comprises a M318V mutation. In some embodiments, a CD117 polypeptide comprises a I319K mutation. In some embodiments, a CD117 polypeptide comprises a V323I mutation. In some embodiments, a CD117 polypeptide comprises a I334V mutation. In some embodiments, a CD117 polypeptide comprises a E360K mutation. In some embodiments, a CD117 polypeptide comprises a P363V mutation. In some embodiments, a CD117 polypeptide comprises a E366D mutation. In some embodiments, a CD117 polypeptide comprises a E376Q mutation. In some embodiments, a CD117 polypeptide comprises a H378R mutation.

[0357] In some embodiments, a CD117 polypeptide comprises one or more mutations that reduce binding of one or more anti-CD117 antibodies or antigen-binding fragments thereof. Various anti-CD117 antibodies or antigen-binding fragments thereof have been described in the art. For example, in some embodiments, an anti-CD117 antibody or antigen-binding fragment thereof comprise or is 2B8, ACK2, AMG191, CDX-0159, Fab79D, FSI-174, or SR-1 or a derivative thereof, or a portion thereof.

[0358] A CD117 polypeptide may comprise one or more mutations to disrupt binding of one or more anti-CD117 antibodies or antigen-binding fragments thereof in regions that do not disrupt one or more wild- type functions, e.g., binding of SCF, of the CD117 polypeptide. In some embodiments, a CD117 polypeptide comprises one or more mutations that do not disrupt binding of SCF. In some embodiments, a CD117 polypeptide comprises one or more mutations and has comparable SCF-binding as a wild-type CD117 polypeptide. In some embodiments, a CD117 polypeptide comprises one or more mutations that reduce binding of one or more anti-CD117 antibodies or antigen-binding fragments thereof.

[0359] In some embodiments, a selection cassette comprises a nucleic acid sequence encoding a base editing system, e.g., as described herein. In some embodiments, a selection cassette comprises a nucleic acid sequence encoding a base editing enzyme and / or a gRNA for a base editing system. In some embodiments, a selection marker comprises a nucleic acid sequence encoding a base editing system targeted to CD117. In some embodiments, a selection cassette comprises a nucleic acid sequence encoding a base editing enzyme and / or a gRNA for a base editing system targeted to CD117. A base editing system can be used to introduce one or more mutations, e.g., one or more mutations described herein, to an endogenousCD117 gene to allow for usage of a CD117 antibody or antigen-binding fragment thereof in enrichment of modified target cells, e.g., target cells comprising a selection cassette integrated into the target cell genomes. Various base editing systems, base editing enzymes, and gRNAs for a base editing system targeted to CD117 have been described in WO 2021 / 041945, which is hereby incorporated by reference in its entirety. Table 12: Exemplary Selection Marker Sequences

[0360] Various promoters can be included in a selection cassette to drive expression of a nucleic acid sequence encoding a selection marker in target cells. A promoter may be a ubiquitous promoter that drives expression of an operably-linked sequence, e.g., a sequence encoding a selection marker in a cell regardless of cell type or a promoter may be a lineage-specific promoter that drives expression of an operably-linked sequence, e.g., a sequence encoding a selection marker in a cell of a specific lineage. In some embodiments, a promoter operably linked to a nucleic acid sequence encoding a selection marker is a ubiquitous promoter. In some embodiments, a promoter operably linked to a nucleic acid sequence encoding a selection marker is a lineage-specific promoter.

[0361] In some embodiments, a selection cassette comprises a ubiquitous promoter. In someembodiments, a selection cassette comprises a ubiquitous promoter operably linked to a selection marker. In some embodiments, a selection cassette comprises an EF1a promoter. In some embodiments, a selection cassette comprises an EF1a promoter comprising a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 107, 108, or 109. In some embodiments, a selection cassette comprises an EF1a promoter comprising a sequence of SEQ ID NO: 107, 108, or 109. In some embodiments, a selection cassette comprises an EF1a promoter that has a nucleic acid sequence of SEQ ID NO: 107, 108, or 109.

[0362] Various polyadenylation (polyA) signal sequences are known in the art and can be used in a selection cassette provided herein. For example, in some embodiments, a polyA signal sequence comprises or is a bGH, hGH, hBG, or SV40 polyA signal sequence. In some embodiments, a polyA signal sequence is located downstream of a nucleic acid sequence encoding a selection marker. In some embodiments, a polyA signal sequence is located in a 3’ UTR of a selection cassette.

[0363] In some embodiments, a selection cassette comprises an SV40 polyA signal sequence. In some embodiments, a selection cassette comprises a polyA signal sequence, e.g., an SV40 polyA signal sequence, that comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 132 or 174. In some embodiments, a selection cassette comprises a polyA signal sequence, e.g., an SV40 polyA signal sequence that comprises a sequence of SEQ ID NO: 132 or 174. In some embodiments, a selection cassette comprises a polyA signal sequence, e.g., an SV40 polyA signal sequence that has a nucleic acid sequence of SEQ ID NO: 132 or 174.

[0364] In some embodiments, a selection cassette comprises a sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 83. In some embodiments, a selection cassette comprises a sequence of SEQ ID NO: 83. In some embodiments, a selection cassette has a nucleic acid sequence of SEQ ID NO: 83. Table 13: Exemplary Selection Cassette SequencesTransposase target sites

[0365] Transposases can include integrases from retrotransposons or integrases of retroviral origin, as well as any protein that is a component of a functional nucleic acid-protein complex capable of transposition. A transposition reaction includes a transposon and a transposase. Transposons are present within payload vectors of the present disclosure and include transposase target sites upstream and downstream of a larger segment of DNA. Additional information on transposons can be found, for instance, in Muñoz-López and García Pérez, Curr Genomics 2010;11(2):115. Transposases bind the transposase target sites and catalyze the movement and integration of the transposon into the genome in a cell of the subject. Transposase target sites may, in some instances, also be referred to as terminal inverted repeats (TIRs).

[0366] In some embodiments, a transposon comprises one or more payload cassettes and / or one or more selection cassettes. In some embodiments, a transposon comprise one or more payload cassettes and one or more selection cassettes. In some embodiments a payload vector comprises one or more payload cassettes and / or one or more selection cassettes between a first transposase target site and a second transposase target site. In some embodiments, a payload vector comprises one or more payload cassettes and one or more selection cassettes between a first transposase target site and a second transposase target site.

[0367] In some embodiments, a transposase target site comprises or is a Sleeping Beauty (SB) transposase target site. In some embodiments, a payload vector or payload vector genome comprises a pair of SB transposase target sites. In some embodiments, a first transposase target site comprises or is a SB transposase target site and a second transposase target site comprises or is a SB transposase target site.

[0368] In some embodiments, a transposase target site comprises a sequence of SEQ ID NO: 84. In some embodiments, a transposase target site has a nucleic acid sequence of SEQ ID NO: 84. In some embodiments, a transposase target site comprises a sequence of SEQ ID NO: 85. In some embodiments, a transposase target site has a nucleic acid sequence of SEQ ID NO: 85.

[0369] In some embodiments, a payload vector comprises a first transposase target site and a second transpose site and the first transposase target site comprises a sequence of SEQ ID NO: 84 and the second transposase target site comprises a sequence of SEQ ID NO: 85. In some embodiments, a payload vector comprises a first transposase target site and a second transpose site and the first transposase target site has a nucleic acid sequence of SEQ ID NO: 84 and the second transposase target site has a nucleic acid sequence of SEQ ID NO: 85. In some embodiments, a payload vector comprises a first transposase target site and asecond transpose site and the first transposase target site comprises a sequence of SEQ ID NO: 85 and the second transposase target site comprises a sequence of SEQ ID NO: 84. In some embodiments, a payload vector comprises a first transposase target site and a second transpose site and the first transposase target site has a nucleic acid sequence of SEQ ID NO: 85 and the second transposase target site has a nucleic acid sequence of SEQ ID NO: 84.

[0370] In some embodiments, a transposase target site comprises or is a PiggyBac (PB) transposase target site. In some embodiments, a payload vector or payload vector genome comprises a pair of PB transposase target sites. In some embodiments, a first transposase target site comprises or is a PB transposase target site and a second transposase target site comprises or is a PB transposase target site.

[0371] In some embodiments, a transposase target site comprises or is a Tol2 transposase target site. In some embodiments, a payload vector or payload vector genome comprises a pair of Tol2 transposase target sites. In some embodiments, a first transposase target site comprises or is a Tol2 transposase target site and a second transposase target site comprises or is a Tol2 transposase target site.

[0372] As those skilled in the art will appreciate, transposase target sites in a payload vector may be chosen in combination with an integration vector to be used to ensure that the transposase target sites included in the payload vector correspond to a transposase encoded by the integration vector. For example, in some embodiments, a payload vector may comprise SB transposase target sites and an integration vector to be used with the payload vector, e.g., in a method of in vivo gene therapy, may comprise a nucleic acid sequence encoding a SB transposase. Table 14: Exemplary Transposase Target Site SequencesRecombinase target sites

[0373] Payload vectors of the present disclosure may comprise recombinase target sites to facilitate higher rates of integration of a payload cassette and / or a selection cassette into a genome of a target cell. In some embodiments, transposons, particularly larger transposons, are most efficiently transposed when present in circularized nucleic acid molecule...

Claims

1. CLAIMS 1. A method of in vivo gene therapy in a subject, comprising administering to the subject a dose of a pharmaceutical composition comprising a helper dependent adenoviral (HDAd) payload vector and a helper dependent adenoviral (HDAd) integration vector, wherein the HDAd payload vector and HDAd integration vector are at a ratio of about 1-3:1 or 1:1-3 HDAd payload vector:HDAd integration vector; wherein the dose is about 2.5 x 1011GC / kg to about 1.25 x 1013GC / kg; wherein the HDAd payload vector comprises: (A) a CD46-binding adenoviral capsid; and (B) a double-stranded DNA genome comprising: (1) a 5’ ITR and a 3’ ITR; (2) a packaging sequence; (3) a first transposase target site and a second transposase target site; (4) a payload cassette comprising: (a) a therapeutic sequence; and (b) a promoter operably linked to the therapeutic sequence; and (5) a selection cassette comprising: (a) a nucleic acid sequence encoding a selection marker; and (b) a promoter operably linked to the nucleic acid sequence encoding the selection marker; wherein: (i) the payload cassette and the selection cassette are located between the first transposase target site and the second transposase target site; and (ii) the packaging sequence is located 5’ to the first transposase target site; and wherein the HDAd integration vector comprises: (A) a CD46-binding adenoviral capsid; and (B) a double-stranded DNA genome comprising: (1) a 5’ ITR and a 3’ ITR; (2) a packaging sequence; and (3) a nucleic acid sequence encoding a transposase.

2. The method of claim 1, wherein the subject is suffering from a condition, disorder, or disease.

3. The method of claim 2, wherein the method comprises treating the condition, disorder, or disease.

4. The method of claim 2 or 3, wherein the condition, disorder, or disease is an immune deficiency disorder.

5. The method of any one of claims 2-4, wherein the subject is suffering from a defect in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex.

6. The method of any one of claims 2-5, wherein the condition, disorder, or disease is chronic granulomatous disease, X-linked chronic granulomatous disease, and / or autosomal recessive chronic granulomatous disease.

7. The method of any one of claims 1-6, wherein: (i) the CD46-binding adenoviral capsid of the HDAd payload vector is an Ad5 / 35 capsid or an Ad5 / 35++ capsid; and / or (ii) the CD46-binding adenoviral capsid of the HDAd integration vector is an Ad5 / 35 capsid or an Ad5 / 35++ capsid.

8. The method of any one of claims 1-7, wherein the therapeutic sequence comprises or is a nucleic acid sequence encoding a therapeutic polypeptide.

9. The method of claim 8, wherein the therapeutic polypeptide comprises or is a CYBB, CYBA, NCF1, NCF2, NCF4, or CYBC1 therapeutic polypeptide.

10. The method of claim 8 or 9, wherein the nucleic acid sequence encoding a therapeutic polypeptide is codon-optimized.

11. The method of any one of claims 8-10, wherein the nucleic acid sequence encoding a therapeutic polypeptide comprises a nucleic acid sequence that has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to the nucleic acid sequence of SEQ ID NO: 79; or wherein the nucleic acid sequence encoding a therapeutic polypeptide comprises or is the nucleic acid sequence of SEQ ID NO:

79.

12. The method of any one of claims 8-11, wherein the therapeutic polypeptide comprises or is the amino acid sequence of any one of SEQ ID NOs: 151-161.

13. The method of any one of claims 1-12, wherein: (i) the 5’ ITR of the genome of the HDAd payload vector is an Ad55’ ITR and the 3’ ITR of the genome of the HDAd payload vector is an Ad53’ ITR; and / or (ii) the packaging sequence of the genome of the HDAd payload vector is an Ad5 packaging sequence.

14. The method of any one of claims 1-13, wherein: (i) the promoter operably linked to the therapeutic sequence comprises or is a lineage-specific promoter;(ii) the promoter operably linked to the therapeutic sequence comprises or is a CTSG-FES promoter; and / or (iii) the promoter operably linked to the therapeutic sequence comprises or is the nucleic acid sequence of any one of SEQ ID NOs: 88-106 and 175.

15. The method of any one of claims 1-14, wherein the payload cassette comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of SEQ ID NO: 80; or the payload cassette comprises or is the nucleic acid sequence of SEQ ID NO:

80.

16. The method of any one of claims 1-15, wherein the genome of the HDAd payload vector comprises a chromatin insulator, optionally wherein the chromatin insulator comprises or is the nucleic acid sequence of SEQ ID NO:

133.

17. The method of any one of claims 1-16, wherein the selection marker is MGMTP140K, optionally wherein the nucleic acid sequence encoding a selection marker comprises or is the nucleic acid sequence of SEQ ID NO:

81.

18. The method of any one of claims 1-16, wherein the selection marker is a CD117 polypeptide comprising one or more mutations.

19. The method of any one of claims 1-18, wherein: (i) the promoter operably linked to the nucleic acid sequence encoding the selection marker comprises or is a ubiquitous promoter; (ii) the promoter operably linked to the nucleic acid sequence encoding the selection marker comprises or is an EF1a, ACTB, EFS, eIF4A1, CAG, CMV, GAPDH, KIN, PGK, ROSA, SFFV, SV40, UBB, or UBC promoter; (iii) the promoter operably linked to the nucleic acid sequence encoding the selection marker comprises is a EF1a promoter; and / or (iv) the promoter operably linked to the nucleic acid sequence encoding the selection marker comprises or is the nucleic acid sequence of any one of SEQ ID NOs: 107-127, 171, and 172.

20. The method of any one of claims 1-19, wherein the selection cassette comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of SEQ ID NO: 83; or the selection cassette comprises or is the nucleic acid sequence of SEQ ID NO:

83.

21. The method of any one of claims 1-20, wherein the first transposase target site is a Sleeping Beauty (SB) transposase target site and the second transposase target site is a SB transposase target site.

22. The method of any one of claims 1-21, wherein the genome of the HDAd payload vector comprises a first recombinase target site and a second recombinase target site, wherein the firsttransposase target site and the second transposase target site are located between the first recombinase target site and the second recombinase target site and the packaging sequence is located 5’ to the first recombinase target site.

23. The method of claim 22, wherein: (i) the first recombinase target site is a Flp recombinase target site and the second recombinase target site is a Flp recombinase target site; and / or (ii) wherein the first recombinase target site is a Flpx9 or FlpE recombinase target site and the second recombinase target site is a Flpx9 or FlpE recombinase target site.

24. The method of any one claims 1-23, wherein the genome of the HDAd payload vector comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of SEQ ID NO: 134; or wherein the genome of the HDAd payload vector comprises or is the nucleic acid sequence of SEQ ID NO:

134.

25. The method of any one claims 1-24, wherein: (i) the transposase comprises or is a Sleeping Beauty (SB) transposase, optionally wherein the SB transposase is a SB10 transposase, SB100X transposase, SB100X+ transposase, or M3A transposase or a variant thereof; and / or (ii) the transposase comprises or is the amino acid sequence of any one of SEQ ID NOs: 135-138.

26. The method of any one of claims 1-25, wherein the genome of the HDAd integration vector comprises a nucleic acid sequence encoding a recombinase.

27. The method of claim 26, wherein: (i) the recombinase comprises or is a Flp recombinase, optionally wherein the Flp recombinase comprises one or more mutations, optionally wherein one or more mutations are selected from P2S, L33S, Y108N, S294P, or a combination thereof; (ii) the recombinase comprises or is a Flpx9 recombinase, FlpE recombinase, or FlpO recombinase; and / or (iii) the recombinase comprises or is the amino acid sequence of any one of SEQ ID NOs: 139- 141.

28. The method of any one of claims 1-27, wherein: (i) the 5’ ITR of the genome of the HDAd integration vector comprises or is an Ad55’ ITR and the 3’ ITR of the genome of the HDAd integration vector comprises or is an Ad53’ ITR; and / or (ii) the packaging sequence of the genome of the HDAd integration vector comprises or is an Ad5 packaging sequence.

29. The method of any one of claims 1-28, wherein the genome of the HDAd integration vector comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of any one of SEQ ID NOs: 144- 146; or wherein the genome of the HDAd integration vector comprises or is the nucleic acid sequence of any one of SEQ ID NOs: 144-146.

30. The method of any one of claims 1-29, wherein the pharmaceutical composition is a liquid composition, optionally wherein the pharmaceutical composition comprises a buffer.

31. The method of any one of claims 1-30, wherein the HDAd payload vector and HDAd integration vector are at a ratio of about 1:1 HDAd payload vector:HDAd integration vector.

32. The method of any one of claims 1-31, wherein the pharmaceutical composition is administered intravenously.

33. The method of any one of claims 1-32, wherein no more than one dose of the pharmaceutical composition is administered to the subject.

34. The method of any one of claims 1-33, wherein the dose is about 2.2 x 1012GC / kg to about 6.25 x 1012GC / kg.

35. The method of any one of claims 1-34, wherein the method comprises mobilizing hematopoietic stem cells of the subject prior to administering the HDAd payload vector.

36. The method of claim 35, wherein mobilizing hematopoietic stem cells of the subject comprises administering to the subject granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), plerixafor, motixafortide, stem cell factor (SCF), tGRO-beta, and / or a VLA4-targeting agent; optionally wherein G-CSF and plerixafor, tGRO-beta and plerixafor, G-CSF and motixafortide, or tGRO-beta and motixafortide are administered to the subject.

37. The method of claim 35 or 36, wherein: (i) G-CSF is administered to the subject daily; (ii) G-CSF is administered to the subject for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days; (iii) G-CSF is administered to the subject for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days prior to administration of the HDAd payload vector; and / or (iv) G-CSF is administered to the subject on the day of administration of the HDAd payload vector.

38. The method of any one of claims 35-37, wherein: (i) plerixafor is administered to the subject daily;(ii) plerixafor is administered to the subject for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days; (iii) plerixafor is administered to the subject for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days prior to administration of the HDAd payload vector; and / or (iv) plerixafor is administered to the subject on the day of administration of the HDAd payload vector.

39. The method of any one of claims 1-38, wherein the method comprises administering to the subject an immunosuppression regimen comprising one or more immunosuppression agents.

40. The method of claim 39, wherein the immunosuppression regimen comprises a corticosteroid, optionally wherein the corticosteroid comprises or is a glucocorticoid, optionally wherein the glucocorticoid comprises or is dexamethasone, prednisone, prednisolone, or methylprednisolone.

41. The method of claim 40, wherein: (i) the corticosteroid is administered to the subject daily; (ii) the corticosteroid is administered to the subject for at least about 1, 2, 3, 4, 5, 6, 7 or more days or at least about 1, 2 or more weeks; (iii) the corticosteroid is administered to the subject for at least about 1, 2, 3, 4, 5 or more days prior to administration of the HDAd payload vector; and / or (iv) the corticosteroid is administered to the subject on the day of administration of the HDAd payload vector.

42. The method of any one of claims 39-41, wherein the immunosuppression regimen comprises an inflammatory signal inhibitor, optionally wherein the inflammatory signal inhibitor comprises or is an interleukin-1 signal inhibitor, optionally wherein the interleukin-1 signal inhibitor comprises or is an interleukin-1 receptor antagonist, optionally wherein the interleukin-1 receptor antagonist comprises or is anakinra.

43. The method of claim 42, wherein: (i) the inflammatory signal inhibitor is administered to the subject daily; (ii) the inflammatory signal inhibitor is administered to the subject for at least about 1, 2, 3, 4, 5 or more days; (iii) the inflammatory signal inhibitor is administered to the subject for at least about 1, 2, 3, 4, 5 or more days prior to administration of the HDAd payload vector; and / or (iv) the inflammatory signal inhibitor is administered to the subject on the day of administration of the HDAd payload vector.

44. The method of any one of claims 39-43, wherein the immunosuppression regimen comprises an interleukin-6 receptor antagonist, optionally wherein the interleukin-6 receptor antagonist comprises or is tocilizumab or siltuximab.

45. The method of claim 44, wherein: (i) the interleukin-6 receptor antagonist is administered to the subject daily; (ii) the interleukin-6 receptor antagonist is administered to the subject for at least about 1, 2, 3, 4, 5 or more days; (iii) the interleukin-6 receptor antagonist is administered to the subject for at least about 1, 2, 3, 4, 5 or more days prior to administration of the HDAd payload vector; and / or (iv) the interleukin-6 receptor antagonist is administered to the subject on the day of administration of the HDAd payload vector.

46. The method of any one of claims 39-45, wherein the immunosuppression regimen comprises an anti-IL-17 antibody, an anti-IL-23 antibody, an anti-TNFa antibody, and / or an anti-IFNg antibody, optionally wherein the anti-IFNg antibody comprises or is emapalumab.

47. The method of any one of claims 1-46, wherein the method comprises administering to the subject one or more selection agents.

48. The method of claim 47, wherein the selection marker is MGMTP140Kand the one or more selection agents comprise: (a) temozolomide (TMZ); (b) O6-benzylguanine (O6BG); and / or (c) 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU).

49. The method of claim 48, wherein: (i) TMZ is administered to the subject orally or intravenously; (ii) TMZ is administered to the subject about once every 28 days or about monthly; (iii) 1, 2, 3, 4, 5 or more doses of TMZ are administered to the subject; (iv) a first dose of TMZ is administered to the subject at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks following administration of the HDAd payload vector; and / or (v) TMZ is administered to the subject at a dose of about 300-600 mg / m2.

50. The method of claim 48 or 49, wherein: (i) O6BG is administered to the subject intravenously; (ii) O6BG is administered to the subject as an intravenous bolus and / or intravenous infusion, optionally wherein the intravenous bolus is administered prior to the intravenous infusion; (iii) O6BG is administered to the subject about once every 28 days or about monthly; (iv) 1, 2, 3, 4, 5 or more doses of O6BG are administered to the subject;(v) a first dose of O6BG is administered to the subject at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks following administration of the HDAd payload vector; (vi) O6BG is administered to the subject at a dose of about 80-280 mg / m2; and / or (vii) a dose of O6BG comprises an intravenous bolus dose of about 70-170 mg / m2O6BG and / or an intravenous infusion dose of about 10-110 mg / m2.

51. The method of any one of claims 48-50, wherein: (i) BCNU is administered to the subject intravenously; (ii) BCNU is administered to the subject about once every 28 days or about monthly; (iii) 1, 2, 3, 4, 5 or more doses of BCNU are administered to the subject; (iv) a first dose of BCNU is administered to the subject at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks following administration of the HDAd payload vector; and / or (v) BCNU is administered to the subject at a dose of about 100-500 mg / m2.

52. The method of any one of claims 48-51, wherein TMZ and O6BG, BCNU and O6BG, or TMZ and BCNU are administered to the subject.

53. The method of claim 47, wherein the selection marker is CD117 comprising one or more mutations and the one or more selection agents comprise an anti-CD117 antibody or antigen binding fragment thereof, optionally wherein the anti-CD117 antibody or antigen binding fragment thereof selectively binds to CD117 not comprising the one or more mutations.

54. The method of any one of claims 1-53, wherein the subject is: (i) a mammal; (ii) a human; (iii) a patient; (iv) an adult or a child; and / or (v) a male or a female.

55. The method of any one of claims 1-54, wherein the subject comprises one or more mutations of a CYBB, CYBA, NCF1, NCF2, NCF4, and / or CYBC1 gene.

56. The method of claim 55, wherein the one or more mutations comprise a point mutation, missense mutation, nonsense mutation, deletion, and / or frameshift mutation.

57. The method of any one of claims 2-56, wherein a symptom of the condition, disorder, or disease is reduced.

58. The method of claim 57, wherein a symptom of the condition, disorder, or disease comprises or is an abscess, a granuloma, a bacterial or fungal infection (optionally wherein the infection is recurrent), abdominal pain (optionally wherein the abdominal pain is chronic), liver dysfunction, lung dysfunctionautoimmune condition (optionally wherein the autoimmune condition comprises or is inflammatory bowel disease, autoimmune pulmonary disease, or lupus), or lymph node swelling.

59. The method of any one of claims 2-58, wherein one or more clinical assessments of the subject are independently improved.

60. The method of claim 59, wherein a clinical assessment is a level of NADPH oxidase activity in a sample from the subject and an improvement is an increase as compared to baseline, optionally wherein: (i) the level of NADPH oxidase activity is assessed in the sample from the subject by a nitroblue tetrazolium assay or dihydrorhodamine flow cytometry assay; and / or (ii) baseline is a level of NADPH oxidase activity in a sample from the subject prior to administration of the HDAd payload vector.

61. The method of claim 59, wherein a clinical assessment is a level of DHR+ neutrophils in a sample from the subject and an improvement is an increase as compared to baseline, optionally wherein: (i) the level of DHR+ neutrophils is assessed in the sample from the subject by a dihydrorhodamine flow cytometry assay; (ii) the level of DHR+ neutrophils is increased by at least about 1-100%, 1-75%, 1-50%, 1-25%, 5-100%, 5-75%, 5-50%, 5-25%, 5-20%, 10-100%, 10-75%, 10-50%, 10-25%, 10-20% or more as compared to baseline; or the level of DHR+ neutrophils is increased by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more as compared to baseline; and / or (iii) baseline is a level of DHR+ neutrophils in a sample from the subject prior to administration of the HDAd payload vector.

62. The method of claim 59, wherein a clinical assessment is a level of expression of the therapeutic polypeptide in a sample from the subject and an improvement is an increase as compared to baseline, optionally wherein: (i) the level of expression of the therapeutic polypeptide is assessed in the sample from the subject by flow cytometry; and / or (ii) baseline is a level of expression of the therapeutic polypeptide in a sample from the subject prior to administration of the HDAd payload vector.

63. A helper dependent adenoviral (HDAd) payload vector comprising: (A) a CD46-binding adenoviral capsid; and (B) a double-stranded DNA genome comprising: (1) a 5’ ITR and a 3’ ITR; (2) a packaging sequence;(3) a first transposase target site and a second transposase target site; (4) a payload cassette comprising: (a) a therapeutic sequence comprising a nucleic acid sequence encoding a therapeutic polypeptide, wherein the therapeutic polypeptides is a CYBB, CYBA, NCF1, NCF2, NCF4, or CYBC1 therapeutic polypeptide; and (b) a promoter operably linked to the therapeutic sequence; and (5) a selection cassette comprising: (a) a nucleic acid sequence encoding a selection marker; and (b) a promoter operably linked to the nucleic acid sequence encoding the selection marker; wherein: (i) the payload cassette and the selection cassette are located between the first transposase target site and the second transposase target site; and (ii) the packaging sequence is located 5’ to the first transposase target site.

64. The HDAd payload vector of claim 63, wherein the CD46-binding adenoviral capsid of the HDAd payload vector is an Ad5 / 35 capsid or an Ad5 / 35++ capsid.

65. The HDAd payload vector of claim 63 or 64, wherein the therapeutic polypeptide comprises or is a CYBB, CYBA, NCF1, NCF2, NCF4, or CYBC1 therapeutic polypeptide.

66. The HDAd payload vector of any one of claims 63-65, wherein the nucleic acid sequence encoding a therapeutic polypeptide is codon-optimized.

67. The HDAd payload vector of any one of claims 63-66, wherein the nucleic acid sequence encoding a therapeutic polypeptide comprises a nucleic acid sequence that has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of SEQ ID NO: 79; or wherein the nucleic acid sequence encoding a therapeutic polypeptide comprises or is the nucleic acid sequence of SEQ ID NO:

79.

68. The HDAd payload vector of any one of claims 63-67, wherein the therapeutic polypeptide comprises or is the amino acid sequence of any one of SEQ ID NOs: 151-161.

69. The HDAd payload vector of any one of claims 63-68, wherein: (i) the 5’ ITR is an Ad55’ ITR and the 3’ ITR is an Ad53’ ITR; and / or (ii) the packaging sequence is an Ad5 packaging sequence.

70. The HDAd payload vector of any one of claims 63-69, wherein: (i) the promoter operably linked to the therapeutic sequence comprises or is a lineage-specific promoter;(ii) the promoter operably linked to the therapeutic sequence comprises or is a CTSG-FES promoter; and / or (iii) the promoter operably linked to the therapeutic sequence comprises or is the nucleic acid sequence of any one of SEQ ID NOs: 88-106 and 175.

71. The HDAd payload vector of any one of claims 63-70, wherein the payload cassette comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of SEQ ID NO: 80; or wherein the payload cassette comprises or is the nucleic acid sequence of SEQ ID NO:

80.

72. The HDAd payload vector of any one of claims 63-71, wherein the genome comprises a chromatin insulator, optionally wherein the chromatin insulator comprises or is the nucleic acid sequence of SEQ ID NO:

133.

73. The HDAd payload vector of any one of claims 63-72, wherein the selection marker is MGMTP140K, optionally wherein the nucleic acid sequence encoding a selection marker comprises or is the nucleic acid sequence of SEQ ID NO:

81.

74. The HDAd payload vector of any one of claims 63-72, wherein the selection marker is CD117 comprising one or more mutations.

75. The HDAd payload vector of any one of claims 63-74, wherein: (i) the promoter operably linked to the nucleic acid sequence encoding the selection marker comprises or is a ubiquitous promoter; (ii) the promoter operably linked to the nucleic acid sequence encoding the selection marker comprises or is a EF1a, ACTB, EFS, eIF4A1, CAG, CMV, GAPDH, KIN, PGK, ROSA, SFFV, SV40, UBB, or UBC promoter; (iii) the promoter operably linked to the nucleic acid sequence encoding the selection marker comprises or is a EF1a promoter; and / or (iv) the promoter operably linked to the nucleic acid sequence encoding the selection marker comprises or is the nucleic acid sequence of any one of SEQ ID NOs: 107-127, 171, and 172.

76. The HDAd payload vector of any one of claims 63-75, wherein the first transposase target site is a Sleeping Beauty (SB) transposase target site and the second transposase target site is a SB transposase target site.

77. The HDAd payload vector of any one of claims 63-76, wherein the genome of the HDAd payload vector comprises a first recombinase target site and a second recombinase target site, wherein the first transposase target site and the second transposase target site are located between the first recombinase target site and the second recombinase target site and the packaging sequence is located 5’ to the first recombinase target site.

78. The HDAd payload vector of claim 77, wherein: (i) the first recombinase target site is a Flp recombinase target site and the second recombinase target site is a Flp recombinase target site; and / or (ii) the first recombinase target site is a Flpx9 or FlpE transposase target site and the second recombinase target site is a Flpx9 or FlpE transposase target site.

79. The HDAd payload vector of any one of claims 63-78, wherein the genome of the HDAd payload vector comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of SEQ ID NO: 134; or the genome of the HDAd payload vector comprises or is the nucleic acid sequence of SEQ ID NO:

134.

80. The HDAd payload vector of any one of claims 63-79, wherein the HDAd payload vector is for use in humans.

81. A cell comprising a HDAd payload vector of any one of claims 63-80, optionally wherein the cell is a hematopoietic stem cell.

82. A pharmaceutical composition comprising a HDAd payload vector of any one of claims 63-80 and one or more pharmaceutically acceptable carriers.

83. The pharmaceutical composition of claim 82, wherein: (i) the pharmaceutical composition comprises a helper dependent adenoviral (HDAd) integration vector; (ii) the pharmaceutical composition is a liquid composition; (iii) the pharmaceutical composition comprises a buffer; (iv) the pharmaceutical composition is formulated for injection to a subject in need thereof; and / or (v) the pharmaceutical composition is formulated for use in humans.

84. A gene therapy system comprising a HDAd payload vector of any one of claims 63-80 and a helper dependent adenoviral (HDAd) integration vector, wherein: the HDAd integration vector comprises: (A) a CD46-binding adenoviral capsid; and (B) a double-stranded DNA genome comprising: (1) a 5’ ITR and a 3’ ITR; (2) a packaging sequence; and (3) a nucleic acid sequence encoding a transposase.

85. The gene therapy system of claim 84, wherein the CD46-binding adenoviral capsid of the HDAd integration vector is an Ad5 / 35 capsid or Ad5 / 35++ capsid.

86. The gene therapy system of claim 84 or 85, wherein: (i) the transposase comprises or is a Sleeping Beauty (SB) transposase, optionally wherein the SB transposase is a SB10, SB100X transposase, SB100X+ transposase, or M3A transposase or a variant thereof; and / or (ii) the transposase comprises or is the amino acid sequence of any one of SEQ ID NOs: 135-138.

87. The gene therapy system of any one of claims 84-86, wherein the genome of the HDAd integration vector comprises a nucleic acid sequence encoding a recombinase.

88. The gene therapy system of claim 87, wherein the recombinase comprises or is a Flp recombinase, optionally wherein: (i) the Flp recombinase comprises one or more mutations, optionally wherein one or more mutations are selected from P2S, L33S, Y108N, S294P, or a combination thereof; (ii) the recombinase comprises or is a Flpx9 recombinase, FlpE recombinase, or FlpO recombinase; and / or (iii) the recombinase comprises or is the amino acid sequence of any one of SEQ ID NOs: 139- 141.

89. The gene therapy system of any one of claims 84-88, wherein: (i) the 5’ ITR of the genome of the HDAd integration vector comprises or is an Ad55’ ITR and the 3’ ITR of the genome of the HDAd integration vector comprises or is an Ad53’ ITR; and / or (ii) the packaging sequence of the genome of the HDAd integration vector comprises or is an Ad5 packaging sequence.

90. The gene therapy system of any one of claims 84-89, wherein the genome of the HDAd integration vector comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of any one of SEQ ID NOs: 144-146; or wherein the genome of the HDAd integration vector comprises or is the nucleic acid sequence of any one of SEQ ID NOs: 144-146.

91. The gene therapy system of any one of claims 84-90, wherein the gene therapy system comprises or is an in vivo gene therapy system.

92. The gene therapy system of any one of claims 84-91, wherein the gene therapy system is for use in humans.

93. A pharmaceutical composition comprising a helper dependent adenoviral (HDAd) payload vector and a helper dependent adenoviral (HDAd) integration vector, wherein the HDAd payload vector and HDAd integration vector are at a ratio of about 1-3:1 or 1:1-3 HDAd payload vector:HDAd integration vector;wherein the HDAd payload vector comprises: (A) a CD46-binding adenoviral capsid; and (B) a double-stranded DNA genome comprising: (1) a 5’ ITR and a 3’ ITR; (2) a packaging sequence; (3) a first recombinase target site and a second recombinase target site; (4) a first transposase target site and a second transposase target site; (5) a payload cassette comprising a nucleic acid sequence of SEQ ID NO: 80; and (6) a selection cassette comprising a nucleic acid sequence of SEQ ID NO: 83; wherein: (i) the first transposase target site and the second transposase target site are located between the first recombinase target site and the second recombinase target site; (ii) the payload cassette and the selection cassette are located between the first transposase target site and the second transposase target site; and (iii) the packaging sequence is located 5’ to the first recombinase target site; and wherein the HDAd integration vector comprises: (A) a CD46-binding adenoviral capsid; and (B) a double-stranded DNA genome comprising: (1) a 5’ ITR and a 3’ ITR; (2) a packaging sequence; (3) a nucleic acid sequence encoding a recombinase; and (4) a nucleic acid sequence encoding a transposase.

94. The pharmaceutical composition of claim 93, wherein: (i) the 5’ ITR of the genome of the HDAd payload vector is an Ad55’ ITR and the 3’ ITR of the genome of the HDAd payload vector is an Ad53’ ITR; (ii) the packaging sequence of the genome of the HDAd payload vector is an Ad5 packaging sequence.

95. The pharmaceutical composition of claim 93 or 94, wherein the genome comprises a chromatin insulator, optionally wherein the chromatin insulator comprises or is the nucleic acid sequence of SEQ ID NO:

133.

96. The pharmaceutical composition of any one of claims 93-95, wherein the first transposase target site is a Sleeping Beauty (SB) transposase target site and the second transposase target site is a SB transposase target site.

97. The pharmaceutical composition of any one of claims 93-96, wherein the first recombinase target site is a Flp recombinase target site and the second recombinase target site is a Flp recombinase target site, optionally wherein the first recombinase target site is a Flpx9 or FlpE transposase target site and the second recombinase target site is a Flpx9 or FlpE transposase target site.

98. The pharmaceutical composition of any one of claims 93-97, wherein the genome of the HDAd payload vector comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of SEQ ID NO: 134; or wherein the genome of the HDAd payload vector comprises or is the nucleic acid sequence of SEQ ID NO:

134.

99. The pharmaceutical composition of any one of claims 93-98, wherein: (i) the 5’ ITR of the genome of the HDAd integration vector comprises or is an Ad55’ ITR and the 3’ ITR of the genome of the HDAd integration vector comprises or is an Ad53’ ITR; (ii) the packaging sequence of the genome of the HDAd integration vector comprises or is an Ad5 packaging sequence.

100. The pharmaceutical composition of any one of claims 93-99, wherein: (i) the transposase comprises or is a Sleeping Beauty (SB) transposase, optionally wherein the SB transposase is a SB10 transposase, SB100X transposase, SB100X+ transposase, or M3A transposase or a variant thereof; and / or (ii) the transposase comprises or is the amino acid sequence of any one of SEQ ID NOs: 135-138.

101. The pharmaceutical composition of claim 93-100, wherein: (i) the recombinase comprises or is a Flp recombinase, optionally wherein the Flp recombinase comprises one or more mutations, optionally wherein one or more mutations are selected from P2S, L33S, Y108N, S294P, or a combination thereof; (ii) the recombinase comprises or is a Flpx9 recombinase, FlpE recombinase, or FlpO recombinase; and / or (iii) the recombinase comprises or is the amino acid sequence of any one of SEQ ID NOs: 139- 141.

102. The pharmaceutical composition of any one of claims 93-101, wherein: (i) the 5’ ITR of the genome of the HDAd integration vector comprises or is an Ad55’ ITR and the 3’ ITR of the genome of the HDAd integration vector comprises or is an Ad53’ ITR; and / or (ii) the packaging sequence of the genome of the HDAd integration vector comprises or is an Ad5 packaging sequence.

103. The pharmaceutical composition of any one of claims 93-102, wherein the genome of the HDAd integration vector comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of any one of SEQ ID NOs: 144-146.

104. The pharmaceutical composition of any one of claims 93-103, wherein: (i) the genome of the HDAd integration vector comprises or is the nucleic acid sequence of SEQ ID NO: 144; (ii) the genome of the HDAd integration vector comprises or is the nucleic acid sequence of SEQ ID NO: 145; or (iii) the genome of the HDAd integration vector comprises or is the nucleic acid sequence of SEQ ID NO:

146.

105. The pharmaceutical composition of any one of claims 93-104, wherein: (i) the pharmaceutical composition is a liquid composition; (ii) the pharmaceutical composition comprises a buffer; and / or (iii) the pharmaceutical composition is formulated for use in humans.

106. A helper dependent (HDAd) integration vector comprising: (A) a CD46-binding adenoviral capsid; and (B) a double-stranded DNA genome comprising: (1) a 5’ ITR and a 3’ ITR; (2) a packaging sequence; (3) a nucleic acid sequence encoding a Flp recombinase comprising the amino acid sequence of SEQ ID NO: 140; (4) a promoter operably linked to the nucleic acid sequence encoding a Flp recombinase and comprising SEQ ID NO: 172; (5) a nucleic acid sequence encoding a Sleeping Beauty (SB) transposase comprising the amino acid sequence of SEQ ID NO: 136; and (6) a promoter operably linked to the nucleic acid sequence encoding a SB transposase and comprising SEQ ID NO:

171.

107. The HDAd integration vector of claim 106, wherein the CD46-binding adenoviral capsid of the HDAd integration vector is an Ad5 / 35 capsid or an Ad5 / 35++ capsid.

108. The HDAd integration vector of claim 106 or 107, wherein: (i) the 5’ ITR of the genome of the HDAd integration vector comprises or is an Ad55’ ITR and the 3’ ITR of the genome of the HDAd integration vector comprises or is an Ad53’ ITR; and / or(ii) the packaging sequence of the genome of the HDAd integration vector comprises or is an Ad5 packaging sequence.

109. The HDAd integration vector of any one of claims 106-108, wherein the genome comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of SEQ ID NO: 145; or wherein the genome comprises or is the nucleic acid sequence of SEQ ID NO:

145.

110. The HDAd integration vector of any one of claims 106-109, wherein the HDAd integration vector is for use in humans.

111. A helper dependent (HDAd) integration vector comprising: (A) a CD46-binding adenoviral capsid; and (B) a double-stranded DNA genome comprising: (1) a 5’ ITR and a 3’ ITR; (2) a packaging sequence; (3) a nucleic acid sequence encoding a Flp recombinase comprising the amino acid sequence of SEQ ID NO: 140; (4) a promoter operably linked to the nucleic acid sequence encoding a Flp recombinase and comprising SEQ ID NO: 171; (5) a nucleic acid sequence encoding a Sleeping Beauty (SB) transposase comprising the amino acid sequence of SEQ ID NO: 136; and (6) a promoter operably linked to the nucleic acid sequence encoding a SB transposase and comprising SEQ ID NO:

172.

112. The HDAd integration vector of claim 111, wherein the CD46-binding adenoviral capsid of the HDAd integration vector is an Ad5 / 35 capsid or an Ad5 / 35++ capsid.

113. The HDAd integration vector of claim 111 or 112, wherein: (i) the 5’ ITR of the genome of the HDAd integration vector comprises or is an Ad55’ ITR and the 3’ ITR of the genome of the HDAd integration vector comprises or is an Ad53’ ITR; and / or (ii) the packaging sequence of the genome of the HDAd integration vector comprises or is an Ad5 packaging sequence.

114. The HDAd integration vector of any one of claims 111-113, wherein the genome comprises a nucleic acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the nucleic acid sequence of SEQ ID NO: 146; or wherein the genome comprises or is the nucleic acid sequence of SEQ ID NO:

146.

115. The HDAd integration vector of any one of claims 111-114, wherein the HDAd integration vector is for use in humans.

116. A vector of any one of claims 63-80 and 106-115, a pharmaceutical composition of any one of claims 82-83 and 93-105, or a gene therapy system of any one of claims 84-92 for use in a method of in vivo gene therapy in a subject.

117. A vector of any one of claims 63-80 and 106-115, a pharmaceutical composition of any one of claims 82-83 and 93-105, or a gene therapy system of any one of claims 84-92 for use in manufacturing a medicament for a method of in vivo gene therapy in a subject.

118. Use of a vector of any one of claims 63-80 and 106-115, a pharmaceutical composition of any one of claims 82-83 and 93-105, or a gene therapy system of any one of claims 84-92 in a method of in vivo gene therapy in a subject.

119. Use of a vector of any one of claims 63-80 and 106-115, a pharmaceutical composition of any one of claims 82-83 and 93-105, or a gene therapy system of any one of claims 84-92 in manufacture of a medicament for a method of in vivo gene therapy in a subject.

120. A method, vector, gene therapy system, pharmaceutical composition, or use of any one of Embodiments 1-267.

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