Compositions and methods for treating CD20 positive central nervous system lymphomas

Abstract

Provided herein is a recombinant adeno-associated virus (rAAV) comprising an AAV capsid and an expression cassette comprising an engineered nucleic acid sequence encoding an anti-hCD20 antibody (i.e., rituximab) comprising heavy chain and light chain operably linked to regulatory control sequences which direct expression of rituximab in a target cell. Also provided are a pharmaceutical composition comprising a rAAV as described herein in a formulation buffer, nucleic acid molecule, packaging host cells, rAAV production system, and a method of treating CD20-positive central nervous system lymphomas

Description

[0001] COMPOSITIONS AND METHODS FOR TREATING CD20 POSITIVE

[0002] CENTRAL NERVOUS SYSTEM LYMPHOMAS

[0003] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0004] The electronic sequence listing filed herewith named “UPN-23-10397PCT.xml” with size of 207,906 bytes, created on date of September 30, 2024, and the contents of the electronic sequence listing (e.g., the sequences and text therein) are incorporated herein by reference in entirety.

[0005] BACKGROUND OF THE INVENTION

[0006] Non-Hodgkin lymphoma (NHL) is the most prevalent type of hematological malignancy, accounting for 4% of all cancer diagnosis in the United States. The American Cancer Society approximates that in 2023, an estimated 80,550 people will be diagnosed with NHL and 20, 180 will die from the disease (cancer.org / cancer / types / non-hodgkin-lymphoma / about / key-statistics). Five-year survival of patients diagnosed with advanced NHL is approximately 64%. NHLs are broadly characterized into B-cell lymphomas and natural killer (NK) / T cell lymphomas: to date, the World Health Organization (WHO) has identified over 60 different subtypes of NHL according to cell of origin, molecular markers, and rate of progression. Average age of diagnosis is 67 years old, but several subtypes of NHL are more common in children and young adults, and immunocompromised patients. NHL usually manifests in the lymph nodes, often spreading to oilier parts of the lymphatic system like spleen and bone marrow .

[0007] Approximately 85% to 90% of NHL originates from B cell precursors and mature B cells. Cluster of differentiation 20 (CD20) antigen is expressed on most B cell NHL. with CD20 negative tumors accounting only for 1% to 2% of all cases of B cell lymphoma. CD20 is a pan B cell surface marker expressed from the pre-B to mature B cell stage and in mature B-cell neoplasms. The biological function of CD20 remains elusive, but recent studies have shown that surface expression of CD20 in mature B cells is important for maintaining stability' of IgM and IgD class of B cell receptor (BCR) containing-protein islands on the surface of resting B cells, preventing uncontrollable activation (Klasener, K. et al, CD20 as a gatekeeper of tire resting state of human B cells. PNAS 2020, 18 (7) e2021342118).

[0008] Rituximab is a chimeric anti-human CD20 monoclonal antibody and the world’s first monoclonal antibody therapy approved for the treatment of oncology patients. Rituximab is widely used in the treatment of NHL since its initial approval by the Food and Drug Administration (FDA) in 1997 to treat relapsed or refractor}', low-grade or follicular, CD20- postive B cell NHL. Addition of rituximab to cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) regimen for the treatment of diffuse large B cell lymphoma (DLBCL), the most common type of aggressive B cell NHL subtype (40-60% of cases), was well tolerated and resulted in a complete response rate of 76% vs. 63% compared to CHOP alone in treatment naive patients (Coiffier B. et al, CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma, NEJM 2002, 346(4):235-242). However, there is a large variation in survival rate among patients according to NHL subtype and stage at diagnosis, and 30-50% of patients will invariably experience disease progression following R- CHOP treatment even after achieving complete remission. An estimated 15% to 25% of patients have primary refractor ’ NHL, failing to respond to therapy or experiencing early relapse.

[0009] As the disease progresses in these subsets of patients, NHL can spread to extra-nodal sites such as the central nervous system (CNS) (secondary CNS lymphoma, SCNSL). or in rare cases, NHL can primarily manifest as a disease confined to the brain, eyes and / or leptomeninges (primary DLBCL of tire CNS. CNS-DLBCL) (Kaji, F. A., et al.. Rare central nervous system lymphomas, British Journal of Haematology, 2022, 197:662-678). CNS-DLBCL accounts for 2- 3% of all NHL cases, while SCNSL arises in approximately 5% of NHL patients. Prognosis of patients with CNS lymphoma are poor, with a median overall survival of 2 months to 13 months. CNS-DLBCL and SCNSL are treated with a combination of non-surgical approaches comprising corticosteroids, systemic and intrathecal chemotherapy, and whole brain radiation therapy. Virtually all patients with CNS lymphoma succumb from the disease.

[0010] Presence of a blood brain barrier (BBB) severely limits the diffusion of large molecules such as therapeutic antibodies into the brain, with only 0. 1% to 0.2% of systemic rituximab reaching the CNS. A series of clinical studies using intrathecal (IT) rituximab therapy as a single agent or combined with a variety of treatments have shown promising results in patients with CNS-DLBCL and SCNSL. A retrospective study in 39 patients (~60 years old) treated twice weekly with 25 mg IT rituximab at the Memorial Sloan Kettering Cancer Center showed a complete response rate of 82% (CNS-DLBCL) and 68% (SCNSL), with an overall survival of 23 and 7 months, respectively. Moreover, 22 / 29 patients with CSF involvement showed a complete cytological response (Umemura & Omuro, Incorporating Intra-Thecal Rituximab into Chemotherapy Regimens for CNS Lymphoma, Neurolog}’ 2016, 86(16)). Use of IT or intraventricular rituximab (10 mg to 40 mg) in 25 children and young adults with CD20-positive CNS lymphoma have shown that 18 / 25 patients achieved CNS remission, including 9 / 10 patients with relapsed or refractory CNS Burkitt NHL or B-cell leukemia and 4 patients with relapsed or refractory post-transplant proliferative disorder (Ceppi, F., et al, Safety and efficacy of intrathecal rituximab in children with B cell lymphoid CD20+ malignancies: An international retrospective study, Am. J. Hematol., 2016, 91: 486-491). Other clinical trials have investigated the benefits of direct CNS delivery of rituximab with similar findings, suggesting that this treatment approach is safe and effective Rubenstein JL, et al., Phase I study of intraventricular administration of rituximab in patients with recurrent CNS and intraocular lymphoma, J Clin Oncol., 2007 Apr 10;25( 11): 1350-6; reviewed in Wyne, A. et al, A Systematic Review on the Use of Intrathecal Rituximab in CNS Lymphoma, Blood 2011, 118(21):4987). Nonetheless, long-term feasibility of this approach is limited by the need for repeated drug administrations to overcome its shorter half-life in the cerebrospinal fluid (CSF) vs. systemic circulation (around 3 hours after 25 mg IT vs. 22 days after intravenous infusion, respectively). Frequent IT or intraventricular drug administration may also carry an increased risk for bacterial infection.

[0011] While current therapies have led to an improved control of the disease, treatment of CD20 Positive CNS Lymphomas is a therapeutic challenge and requires improvement.

[0012] SUMMARY OF THE INVENTION

[0013] In one embodiment, a recombinant adcno-associatcd virus (rAAV) comprising an adcno- associated virus (AAV) capsid and a nucleic acid molecule comprising an expression cassette in the capsid is provided, wherein the expression cassette comprises regulatory sequences operably linked to coding sequences for the anti-human CD20 heavy chain and the anti-human CD20 light chain. In certain embodiments, the expression cassette comprises a UbC promoter, a nucleic acid coding sequence for anti-human CD20 (hCD20) antibody heavy chain having an IL2 leader sequence, at least one linker sequence, a nucleic acid coding sequence for an anti-human CD20 (hCD20) light chain having an IL-2 leader sequence. In certain embodiments, the nucleic acid sequence encoding the anti-hCD20 heavy chain comprises SEQ ID NOs: 5, 1, 3 or 7. In certain embodiments, tire expression cassette further comprises a furin cleavage site and a T2A element linker between the coding sequences for the heavy and light chains. In certain embodiments, the nucleic acid sequence encoding the anti-hCD20 light chain comprising SEQ ID NOs: 6, 2. 4 or 8. In certain embodiments, the expression cassette comprises, in addition to a ubiquitin C (UbC) promoter, an optional enhancer, an intron, and a polyadenylation (polyA) sequence. In certain embodiments, tire nucleic acid molecule comprises an AAV inverted terminal repeat (ITR), the expression cassette, and an AAV ITR. In other embodiments, the AAV 5' ITR is 5' to the 5' end of the expression cassette and the AAV 3' ITR is 3' to the 3' end of the expression cassette. In certain embodiments, the nucleic acid sequence encoding a leader peptide comprises SEQ ID NO: 20. In certain embodiments, tire leader peptide comprises SEQ ID NO: 21. In certain embodiments, tire anti-hCD20 heavy chain coding sequence comprises the nucleic acid sequence of SEQ ID NO: 5, or the sequence at least 99% identical to SEQ ID NO: 5 encoding an amino acid sequence of SEQ ID NO: 9, and wherein the anti-hCD20 light chain coding sequence comprises the nucleic acid sequence of SEQ ID NO: 6 or the sequence at least 99% identical to SEQ ID NO: 6 encoding an amino acid sequence of SEQ ID NO: 10. In certain embodiments, the anti-hCD20 heavy chain coding sequence comprises the nucleic acid sequence of SEQ ID NO: 5, and wherein tire anti-hCD20 light chain coding sequence comprises the nucleic acid sequence of SEQ ID NO: 6. In certain embodiments, tire anti-hCD20 antibody coding sequence comprises the nucleic acid sequence of SEQ ID NO: 19. or the sequence at least 95% identical to SEQ ID NO: 19. In certain embodiments, the anti-hCD20 antibody coding sequence comprises the nucleic acid sequence of SEQ ID NO: 52, the sequence at least 95% identical to SEQ ID NO: 52, SEQ ID NO: 53, the sequence at least 95% identical to SEQ ID NO: 53, SEQ ID NO: 54, or the sequence at least 95% identical to SEQ ID NO: 54. In certain embodiments, the UbC promoter has a nucleic acid sequence of SEQ ID NO: 32. In certain embodiments, the polyA sequence is SV40 polyA sequence having a nucleic acid sequence of SEQ ID NO: 34. In certain embodiments, the intron is a chimeric intron having a nucleic acid sequence of SEQ ID NO: 33. In certain embodiments, the AAV capsid is a clade F AAV capsid. In certain embodiments, the capsid is an AAVhu68 capsid. In certain embodiments, the AAV capsid is a mutant AAV9 capsid. In certain embodiments, the AAV capsid is an AAVrh91 capsid, an AAV9 capsid, a AAVhu95 capsid, an AAVhu96 capsid, or an AAVrh32 / 33 capsid.

[0014] In certain embodiments, tire rAAV is for use in treating one or more of a CD20-positive B-ccll lymphoma, CD20-positivc B-ccll Non-Hodgkins Lymphoma, a CD20-positivc central nervous system (CNS) diffuse large B cell lymphoma (CNS-DLBCL). or CD20-positive B-cell secondary CNS lymphoma (SCNSL). In certain embodiments, tire rAAV is formulated for systemic and / or central nervous system delivery. In certain embodiments, the composition comprises a stock of rAAV encoding the anti- CD20 antibody and an aqueous suspension media. In certain embodiments, the suspension is formulated for intravenous or intrathecal delivery, optionally wherein the intrathecal delivery is an intracerebroventricular (ICV) injection or an intracistemal magna (ICM) injection.

[0015] In certain embodiments, a recombinant nucleic acid molecule comprises nucleic acid sequences comprises a vector genome for packaging into an rAAV capsid comprising: (a) an AAV 5' inverted terminal repeat (ITR), (b) an expression cassette comprising a coding sequence for an anti-human CD20 (hCD20) antibody comprising an anti-hCD20 heavy chain and an anti- hCD20 light chain, and regulatory control sequences which are nucleic acid sequences operably linked to the coding sequence which regulate expression of the anti-hCD20 heavy chain and anti- hCD20 light chain, and (c) an AAV 3' ITR.

[0016] In one embodiment, a packaging host cell for rAAV production outside the human body (e.g., in culture) which comprises a recombinant nucleic acid molecule, rep coding sequences operably linked to sequences which express rep protein in the packaging host cell, an AAV capsid coding sequences operably linked to sequences which express AAV capsid proteins in the packaging host cell, and helper virus functions necessary to permit packaging of the expression cassette and AAV ITRs into the AAV capsid. In certain embodiments, the AAV capsid is an AAV9 capsid, a mutant AAV9 capsid, an AAVrh91 capsid, an AAVhu95 capsid, an AAVrh32 / 33 capsid, or an AAVhu96 capsid. In certain embodiments, an rAAV production is provided which comprises a cell culture which may be adherent or in suspension comprising tire packaging host cell.

[0017] In certain embodiments, a method for treating one or more of a CD20-positive B-cell lymphoma, CD20-positive B-cell Non-Hodgkins Lymphoma, a CD20-positive central nervous system (CNS) diffuse large B cell lymphoma (CNS-DLBCL). or CD20-positive B-cell secondary CNS lymphoma (SCNSL) is provided. The method comprises administering to the subject a suspension comprising the rAAV in a formulation buffer. In a certain embodiment, the suspension is administered intravenously or intrathecally.

[0018] In certain embodiments, an anti-ncoplastic regimen is provided which comprises administering rAAV, a composition, or a pharmaceutical composition as provided herein in combination with a biologic drug, a small molecule, anti-neoplastic agent, radiation, and / or chemotherapeutic agent. These and other aspects of tire invention are apparent from the following detailed description of the invention.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1A shows rituximab expression (micrograms (pg) / ml) in scrum in Ragl KO mice treated by ICV administration of 1 x 1011GC of AAVhu68. Rituximab. coGW with and without IVIG.

[0021] FIG. IB shows rituximab expression (nanograms per mg protein) in perfused brain in Ragl KO mice treated by ICV administration of 1 x 1011GC of AAVhu68. Rituximab. coGW with and without IVIG.

[0022] FIG. 1C shows levels of rituximab DNA, plotted as GC / diploid cell, as measured in brain and liver tissue in Ragl KO mice treated by ICV administration of 1 x 1011GC of AAVhu68.Rituximab.coGW with and without IVIG.

[0023] FIG. ID shows levels of rituximab RNA, plotted as copies per 100 ng RNA1, as measured in brain and liver tissue in Ragl KO mice treated by ICV administration of 1 x 1011GC of AAVhu68.Rituximab.coGW with and without IVIG.

[0024] FIG. 2A shows biodistribution analysis with plotted measurement of DNA concentration (GC / pg) in the collected liver and brain tissue samples following administration with AAV. rituximab. coGA, AAV. rituximab. coGS, AAV. rituximab. coGW, and AAV. rituximab. coNP.

[0025] FIG. 2B shows biodistribution analy sis with plotted measurement of RNA concentration (total RNA copies / lOOng) in the collected liver and brain tissue samples following administration with AAV.rituximab.coGA, AAV.rituximab.coGS, AAV.rituximab.coGW, and AAV.rituximab.coNP.

[0026] FIG. 2C shows results of the ELISA with plotted measurements of rituximab concentration (ng / mg protein) in the perfused brain tissue samples collected post administration with AAV.rituximab.coGA, AAV.rituximab.coGS, AAV.rituximab.coGW, and A A V .rituximab. coNP .

[0027] FIG. 2D shows results of the ELISA with plotted measurements of rituximab concentration (pg / mL) in the serum samples collected at day 0, 7, 14, and 30 post administration with AAV.rituximab.coGA. AAV.rituximab.coGS, AAV.rituximab.coGW, and AAV.rituximab.coNP. FIG. 3 A shows a representative western blot confirming expression of the heavy and light chain of rituximab in brain tissue following administration with AAV.rituximab.coGW.

[0028] FIG. 3B shows a plot of the relative protein level of the heavy and light chain of rituximab expression in brain tissue following administration with AAV.rituximab.coGW.

[0029] FIG. 4A shows quantified results of the tumor burden assessment (bioluminescence assessment via in vivo imaging system (IVIS)) in xenograft mice (Ramos xenograft) post treatment with AAVhu68.Rituximab.coGW in comparison with isotype control.

[0030] FIG. 4B shows Kaplan-Meier survival analysis of probability of survival in tumor bearing mice (Ramos xenograft) treated with AAVhu68. Rituximab. coGW.

[0031] FIG. 5A shows quantified results of the tumor burden assessment (bioluminescence assessment via IVIS) in xenograft mice (OciLy3 xenograft) post treatment with AAVhu68.Rituximab.coGW in comparison with isotype control.

[0032] FIG. 5B shows Kaplan-Meier survival analysis of probability of survival in tumor bearing mice (OciLy3 xenograft) treated with AAVhu68.Rituximab.coGW.

[0033] FIG. 6A shows quantified results of the tumor burden assessment (bioluminescence assessment via IVIS) in xenograft mice (Daudi xenograft) post treatment with AAVhu68.Rituximab.coGW in comparison with isotype control.

[0034] FIG. 6B shows Kaplan-Meier survival analysis of probability of survival in tumor bearing mice (Daudi xenograft) treated with AAVhu68.Rituximab.coGW.

[0035] FIG. 7 shows Kaplan-Meier surv ival analysis of probability of survival in tumor bearing mice (patient-derived xenograft) treated with AAVhu68. Rituximab. coGW.

[0036] DETAILED DESCRIPTION OF THE INVENTION

[0037] Provided herein are recombinant, replication-defective adeno-associated virus (rAAV) vectors having an AAV capsid and a vector genome comprising an expression cassette comprising a nucleic acid sequence encoding a heavy chain and a light chain of anti- human CD20 antibody (e.g., anti-hCD20, rituximab), and compositions containing same which are suitable for administration for treatment of CD20-positive lymphomas. Various methods of administration are provided, including systemic administration (e.g.. intravenous), and administration to the central nervous system (e.g.. intrathecal administration) for treatment of CD20-positive lymphomas. Also provided are pharmaceutical compositions, formulations containing same, and in particularly, an aqueous liquid suspension. Uses of these compositions are also provided. Also provided are method of compositions useful for the treatment and / or prevention of CD20-positive lymphomas in the central nervous system (CNS).

[0038] In certain embodiments, the compositions and regimens described herein are useful for delivery' of anti-hCD20 antibody (i.e., also can referred to throughout as immunoglobulin construct) to the central nervous system (CNS). Compositions described herein comprising AAV-Ig are well suited for central nervous system (CNS) CD20-positive lymphomas. The compositions and regimens are also useful for treating primary and / or secondary CD20-positive lymphomas. Various other indications are described herein.

[0039] The rAAV vectors, as described herein encode the anti-hCD20 antibody (e.g., rituximab) heavy chain and light chain. See, e.g., drugbank. com / drugs / DB00073. The encoded rituximab heavy chain amino acid sequence is: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGD TSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAG TTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 55; drugbank. ca / drugs / DB00073).

[0040] In certain embodiments, tire expression cassette comprises a nucleic acid sequences which encodes an anti-hCD20 heavy chain amino acid sequence is: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGD TSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAG TTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 9; i.e., without terminal lysine (K)). The encoded amino acid sequence of the anti-hCD20 light chain is: QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRF SGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 56 (drugbank_ca / drugs / DB00073), also SEQ ID NO: 10)), which sequences are incorporated herein by reference.

[0041] In the expression cassette provided herein, each of the heavy chain and light chain is provided with an exogenous leader sequence at its N -terminus. Without wishing to be bound by theory, it is believed that the use of the same exogenous leader sequence (e.g., an IL2-leader) facilitates self-assembly of the heavy and light chains which are expressed as separate proteins in the cells. Additional details are provided herein.

[0042] As described herein, a series of novel rAAV.anti-hCD20-antibody (rAAV.rituximab or A AV. rituximab) constructs have been developed which have demonstrated high yield, expression levels, and / or activity. In certain embodiments, the size and identity of the antibody (protein) chains can be confirmed by western blot. In certain embodiments, the size and identity of the antibody (protein) chains can be confirmed by mass spectrometry (e.g., LC-MS).

[0043] Provided herein are coding sequence(s), expression cassette(s), and vector genome(s) encoding the anti-hCD20 heavy chain (HC) amino acid sequence of SEQ ID NO: 9 (amino acids 1 to 450 of SEQ ID NO: 55) and a light chain (LC) amino acid sequence of SEQ ID NO: 10 (also SEQ ID NO: 56), each of which has been engineered to further have an exogenous leader sequence (also referred to as "leader peptide" or ‘"signal peptide’" or “signal sequence”) for each the heavy chain and the light chain (e.g., SEQ ID NOs: 11, 13, 15, 17 for heavy chain amino acid sequence comprising leader peptide, SEQ ID NOs: 12, 14, 16, 18 for light chain amino acid sequence comprising a leader peptide). In certain constructs illustrated herein, the leader sequence is derived from a human interleukin 2 (IL2 or IL-2) leader sequence. In certain embodiments, the IL2 is same for each the heavy chain and the light chain. In certain embodiments, the IL2 is a modified IL2 for at least one of the heavy chain and the light chain. In certain embodiments, the IL2 leader peptide is a modified human IL2 leader for the heavy chain. In certain embodiments, die IL2 leader peptide is a modified human IL2 leader for the light chain. Further, in certain constructs illustrated in the working examples, the heavy and light chains are separated by a T2A linker which may result in one or more extra amino acids being added to the heavy chain [SEQ ID NOs: 1. 3, 5, 7 or SEQ ID NOs: 11, 13, 15, 17 (comprising leader peptide)]. Further, in certain constructs illustrated in the working examples, the heavy and light chains are separated by a furin / T2A linker which may result in one or more extra amino acids being added to the heavy chain [SEQ ID NOs: 1, 3, 5, 7 or SEQ ID NOs: 11, 13, 15, 17 (comprising leader peptide)]. In one embodiment, a single arginine [R] is added to the heavy chain. However, in certain embodiments, another linker may be selected and / or a different system may result in no additional amino acid, or one or more extra amino acids [e.g., R, Lys (K), RK, RKR, RKRR (SEQ ID NO: 35) among others]. In the constructs encoding the anti-hCD20 antibody product, various designation following the term rituximab, e.g., rituximab-coGW. rituximab-coGS. rituximab-coGA, rituximab-coNP, refers to different nucleic acid coding sequences for the anti- hCD20 antibody heavy chain and light chain. It will be understood that the resulting anti-hCD20 antibody product, following cleavage of the leader peptides and assembly of tire anti-CD20 light and heavy chains, optionally, with the mutant amino acid, is referred to herein alternatively as an “anti-hCD20 antibody” or rituximab, but may contain a variant or mutant amino acid sequence as compared to the amino acid sequence of SEQ ID NO: 55 (drugbank.ca / drugs / DB00073; rituximab heavy chain) and SEQ ID NO: 56 (drugbank ca / drugs / DB00073; rituximab light chain). In certain embodiments, anti-hCD20 antibody may comprise one or more conservative, non-conservative amino acid substitutions, as well as insertions, truncations and / or deletions as compared to amino acid sequence of SEQ ID NO: 55 (drugbank.ca / dmgs / DB00073; rituximab heavy chain) and SEQ ID NO: 56 (drugbank_ca / drugs / DB00073; rituximab light chain). In certain embodiments, tire amino acid sequence of the anti-hCD20 antibody comprises a truncation of the terminal Lysine (K) at the C-tenninus of the amino acid sequence as compared to SEQ ID NO: 55.

[0044] In certain embodiments, the encoded amino acid sequence of the anti-hCD20 has 663 amino acids, including anti-hCD20 heavy chain and light chain separated by extra amino acids as a result of the linker. For example, while each of the described herein expression cassettes encodes the same anti-hCD20 heavy chain and light chain, in one embodiment, there may be one amino acid added to the last position of the heavy chain. In still other embodiments, there may be two, three, four or more extra amino acids attached to the heavy chain. For example, in certain embodiments, the nucleic acid sequences coding for the heavy and light chains of ant-hCD20 antibody are separated by a self-cleaving furin / T2A linker. A furin recognition site that consists of arginine-lysine-arginine-arginine amino acid sequence may be used. Due to the mechanism of furin-mediated cleavage, vector-expressed rituximab may contain an additional arginine (R) residue added to the last position of the heavy chain. In other embodiments, the vector-expressed ant-hCD20 is modified to contain the dipeptide arginine-lysine at the end of the heavy chain, the tripeptide arginine-lysine-arginine at the end of the heavy chain, or the polypeptide arginine- lysine-arginine-arginine at the end of the heavy chain. In certain embodiments, the vector- expressed anti-hCD20 immunoglobulins are a heterogeneous mixture of tw o or more of these immunoglobulin products. Ollier furin cleavage sites can be used (arginine-X-X-arginine, or arginine-X-lysine or arginine-arginine), which can also generate C-terminal heterogeneity. In other words, other vector expressed anti-hCD20 immunoglobulins may be a heterogeneous population of the immunoglobulins in which the heavy chain has 0, 1, 2, 3, or 4 amino acids at its C -terminus as a result of the linker processing.

[0045] In addition, the coding sequences for immunoglobulin heavy chain (HC) and light chain (LC) each contain a heterologous leader peptide which directs each of the immunoglobulin chains into appropriate cellular compartment where the leader peptide is processed away from the mature immunoglobulin chain by the host cellular machinery, e.g., HC with leader peptide comprises amino acid sequence of SEQ ID NO: 57, and LC with leader peptide comprises amino acid sequence of SEQ ID NO: 58 and the two chains are permitted to self-assemble in vivo into a recombinant anti-hCD20 antibody.

[0046] In certain embodiments, the self-assemble in vivo into a recombinant anti-hCD20 antibody can be confirmed with native gel electrophoresis (e.g., polyacrylamide gel electrophoresis (native PAGE)) and denaturing gel electrophoresis (i.e., SDS-PAGE). In certain embodiments, the anti-hCD20 antibody comprises no HC or LC leader peptide sequences (HC amino acid of SEQ ID NO: 9 and LC amino acid sequence of SEQ ID NO: 10).

[0047] As used herein, the term “rituximab” refers to an immunoglobulin construct (“anti-hCD20 antibody”) comprising a heavy chain having an amino acid sequence of SEQ ID NO: 9 or a sequence about 95% to about 100% identical thereto, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 99.9% identical thereto, and values therebetween, as determined over contiguous amino acid sequences (e.g., heavy chain has -1, 0, 1, 2, 3, or 4 amino acids at its C- temiinus as a result of the linker processing), and comprising light chain having an amino acid sequence of SEQ ID NO: 10 or a sequence about 99% to about 100% identical thereto, or at least 99.9% identical thereto, and values therebetween (e.g., as a result of linker processing), as determined over contiguous amino acid sequences, which provide at least 10%, at least 20%, at least 30%, at least 40%. at least 50%, at least 60%, at least 70%. at least 75%, at least 80%, at least 90%, or a similar and / or same, or greater than 100% biological activity (e.g.. binding) or function of rituximab immunoglobulin product. In certain embodiments, greater than 100%, e.g., about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, or greater of rituximab as produced in vitro (e.g., in CHO cells) activity' and / or function is achieved. This biological activity or function may be determined by any suitable means, e.g., in an in vitro assay, animal model or by monitoring post-treatment patients. In certain embodiments, the AAV- expressed rituximab immunoglobulin heavy chain and light chain provide advantages over protein-based rituximab compositions derived from in vitro-produced rituximab including, e.g., improved glycosylation, decreased deamidation, and / or improved stability.

[0048] In one embodiment, the engineered coding sequences for the heavy chain and light chain of rituximab are provided in SEQ ID NO: 19. In one embodiment, the engineered coding sequences for tire heavy chain and light chain of rituximab are provided in SEQ ID NO: 52. In one embodiment, the engineered coding sequences for the heavy chain and light chain of rituximab are provided in SEQ ID NO: 53. In one embodiment, the engineered coding sequences for the heavy chain and light chain of rituximab are provided in SEQ ID NO: 54.

[0049] In certain embodiments, provided herein is rituximab engineered coding sequence comprising a nucleic acid sequence encoding a leader peptide operably linked to rituximab heavy chain, a nucleic acid sequence of SEQ ID NO: 5 or a sequence at least 98% identical to SEQ ID NO: 5 encoding a rituximab heavy chain, a linker, a nucleic acid sequence encoding a leader peptide operably linked to a rituximab light chain, and a nucleic acid sequence comprising SEQ ID NO: 6 or a sequence at least 98% identical to SEQ ID NO: 6 encoding a rituximab light chain. In certain embodiments, provided herein is rituximab engineered coding sequence comprising a nucleic acid sequence encoding a leader peptide operably linked to rituximab heavy chain, a nucleic acid sequence of SEQ ID NO: 5 or a sequence at least 99% identical to SEQ ID NO: 5 encoding a rituximab heavy chain, a linker, a nucleic acid sequence encoding a leader peptide operably linked to a rituximab light chain, and a nucleic acid sequence comprising SEQ ID NO: 6 or a sequence at least 99% identical to SEQ ID NO: 6 encoding a rituximab light chain. In certain embodiments, provided herein is rituximab engineered coding sequence comprising a nucleic acid sequence encoding a leader peptide operably linked to rituximab heavy chain, a nucleic acid sequence of SEQ ID NO: 5 encoding a rituximab heavy chain, a linker, a nucleic acid sequence encoding a leader peptide operably linked to a rituximab light chain, and a nucleic acid sequence comprising SEQ ID NO: 6 encoding a rituximab light chain. In certain embodiments, provided herein is rituximab engineered coding sequence comprising a nucleic acid sequence encoding a leader peptide operably linked to rituximab heavy chain, a nucleic acid sequence of SEQ ID NO: 1 encoding a rituximab heavy chain, a linker, a nucleic acid sequence encoding a leader peptide operably linked to a rituximab light chain, and a nucleic acid sequence comprising SEQ ID NO: 2 encoding a rituximab light chain. In certain embodiments, provided herein is rituximab engineered coding sequence comprising a nucleic acid sequence encoding a leader peptide operably linked to rituximab heavy chain, a nucleic acid sequence of SEQ ID NO: 3 encoding a rituximab heavy chain, a linker, a nucleic acid sequence encoding a leader peptide operably linked to a rituximab light chain, and a nucleic acid sequence comprising SEQ ID NO: 4 encoding a rituximab light chain. In certain embodiments, provided herein is rituximab engineered coding sequence comprising a nucleic acid sequence encoding a leader peptide operably linked to rituximab heavy chain, a nucleic acid sequence of SEQ ID NO: 7 encoding a rituximab heavy chain, a linker, a nucleic acid sequence encoding a leader peptide operably linked to a rituximab light chain, and a nucleic acid sequence comprising SEQ ID NO: 8 encoding a rituximab light chain.

[0050] In certain embodiments, the linker between the heavy chain and light chain is a Thosea asigna virus (T2A) linker. In certain embodiments, the T2A linker comprises nucleic acid sequence of SEQ ID NO: 36, or a sequence at least 95% identical to SEQ ID NO: 36. In certain embodiments, the linker further comprises a furin cleavage at 5 ’ end of the T2A, optionally connected via a flexible linker (e.g., “GSG linker”). In certain embodiments, the furin cleavage sequence comprises nucleic acid sequence of SEQ ID NO: 35. or a sequence at least 95% identical to SEQ ID NO: 35. In certain embodiments, the furin / T2A linker comprises nucleic acid sequence of SEQ ID NO: 37, or a sequence at least 95% identical to SEQ ID NO: 37.

[0051] In certain embodiments, provided herein is rituximab engineered coding sequence comprising a nucleic acid sequence encoding a leader peptide operably linked to rituximab heavy chain, a nucleic acid sequence of SEQ ID NO: 5 or a sequence at least 98% identical to SEQ ID NO: 5 encoding a rituximab heavy chain, a furin / T2A linker, a nucleic acid sequence encoding a leader peptide operably linked to a rituximab light chain, and a nucleic acid sequence comprising SEQ ID NO: 6 or a sequence at least 98% identical to SEQ ID NO: 6 encoding a rituximab light chain. In certain embodiments, provided herein is rituximab engineered coding sequence comprising a nucleic acid sequence encoding a leader peptide operably linked to rituximab heavy chain, a nucleic acid sequence of SEQ ID NO: 5 encoding a rituximab heavy chain, a furin / T2A linker, a nucleic acid sequence encoding a leader peptide operably linked to a rituximab light chain, and a nucleic acid sequence comprising SEQ ID NO: 6 encoding a rituximab light chain. In certain embodiments, provided herein is rituximab engineered coding sequence comprising a nucleic acid sequence encoding a leader peptide operably linked to rituximab heavy chain, a nucleic acid sequence of SEQ ID NO: 1 encoding a rituximab heavy chain, a furin / T2A linker, a nucleic acid sequence encoding a leader peptide operably linked to a rituximab light chain, and a nucleic acid sequence comprising SEQ ID NO: 2 encoding a rituximab light chain. In certain embodiments, provided herein is rituximab engineered coding sequence comprising a nucleic acid sequence encoding a leader peptide operably linked to rituximab heavy chain, a nucleic acid sequence of SEQ ID NO: 3 encoding a rituximab heavy chain, a furin / T2A linker, a nucleic acid sequence encoding a leader peptide operably linked to a rituximab light chain, and a nucleic acid sequence comprising SEQ ID NO: 4 encoding a rituximab light chain. In certain embodiments, provided herein is rituximab engineered coding sequence comprising a nucleic acid sequence encoding a leader peptide operably linked to rituximab heavy chain, a nucleic acid sequence of SEQ ID NO: 7 encoding a rituximab heavy chain, a furin / T2A linker, a nucleic acid sequence encoding a leader peptide operably linked to a rituximab light chain, and a nucleic acid sequence comprising SEQ ID NO: 8 encoding a rituximab light chain.

[0052] In certain embodiments, tire rituximab heavy chain has the amino acid sequence of SEQ ID NO: 9 with a leader sequence. In other embodiments, the rituximab light chain has the amino acid sequence of SEQ ID NO: 10 with a leader sequence. For example, the leader sequence may be from about 15 to about 25 amino acids, preferably about 20 amino acids. In certain embodiments, tire processing of rituximab heavy chain and light chain is directed by leader peptides that are derived from human IL2 protein. In one embodiment the leader sequence is an interleukin (IL) IL-2 leader sequence, which may be the wild-type human IL2, MYRMQLLSCIALSLALVTNS [SEQ ID NO: 21], or a mutated leader, such as MYRMQLLLLIALSLALVTNS [SEQ ID NO: 23], In certain embodiments, the leader peptide comprises nucleic acid sequence comprising SEQ ID NO: 20 or a sequence at least 95% identical to SEQ ID NO: 20. In certain embodiments, the leader peptide comprises nucleic acid sequence comprising SEQ ID NO: 20 or a sequence at least 95% identical to SEQ ID NO: 20 encoding SEQ ID NO: 21. In certain embodiments, the leader peptide comprises nucleic acid sequence comprising SEQ ID NO: 22 or a sequence at least 95% identical to SEQ ID NO: 22. In certain embodiments, the leader peptide comprises nucleic acid sequence comprising SEQ ID NO: 22 or a sequence at least 95% identical to SEQ ID NO: 22 encoding SEQ ID NO: 23. In certain embodiments, the processing of rituximab heavy chain and light chain is directed by leader peptides that are same. In certain embodiments, the processing of rituximab heavy chain and light chain is directed by leader peptides that are different.

[0053] In certain embodiments, provided herein is a rituximab engineered coding sequence comprising a nucleic acid sequence comprising SEQ ID NO: 20 encoding a leader peptide operably linked to rituximab heavy chain, a nucleic acid sequence comprising SEQ ID NO: 1 encoding a rituximab heavy chain, a linker comprising a furin cleavage site (SEQ ID NO: 35). “GSG” linker and T2A (SEQ ID NO: 36), wherein furin cleavage site is at 5’ end of the T2A, a nucleic acid sequence comprising SEQ ID NO: 22 encoding a leader peptide operably linked to a rituximab light chain, and a nucleic acid sequence comprising SEQ ID NO: 2 encoding a rituximab light chain.

[0054] In certain embodiments, provided herein is a rituximab engineered coding sequence comprising a nucleic acid sequence comprising SEQ ID NO: 20 encoding a leader peptide operably linked to rituximab heavy chain, a nucleic acid sequence comprising SEQ ID NO: 3 encoding a rituximab heavy chain, a linker comprising a furin cleavage site (SEQ ID NO: 35), “GSG” linker and T2A (SEQ ID NO: 36), wherein furin cleavage site is at 5’ end of the T2A, a nucleic acid sequence comprising SEQ ID NO: 22 encoding a leader peptide operably linked to a rituximab light chain, and a nucleic acid sequence comprising SEQ ID NO: 4 encoding a rituximab light chain.

[0055] In certain embodiments, provided herein is a rituximab engineered coding sequence comprising a nucleic acid sequence comprising SEQ ID NO: 20 encoding a leader peptide operably linked to rituximab heavy chain, a nucleic acid sequence comprising SEQ ID NO: 5 encoding a rituximab heavy chain, a linker comprising a furin cleavage site (SEQ ID NO: 35), “GSG” linker and T2A (SEQ ID NO: 36), wherein furin cleavage site is at 5’ end of the T2A, a nucleic acid sequence comprising SEQ ID NO: 22 encoding a leader peptide operably linked to a rituximab light chain, and a nucleic acid sequence comprising SEQ ID NO: 6 encoding a rituximab light chain.

[0056] In certain embodiments, provided herein is a rituximab engineered coding sequence comprising a nucleic acid sequence comprising SEQ ID NO: 20 encoding a leader peptide operably linked to rituximab heavy chain, a nucleic acid sequence comprising SEQ ID NO: 7 encoding a rituximab heavy chain, a linker comprising a furin cleavage site (SEQ ID NO: 35), “GSG” linker and T2A (SEQ ID NO: 36), wherein furin cleavage site is at 5’ end of the T2A, a nucleic acid sequence comprising SEQ ID NO: 22 encoding a leader peptide operably linked to a rituximab light chain, and a nucleic acid sequence comprising SEQ ID NO: 8 encoding a rituximab light chain.

[0057] As used herein, the term “biological sample'’ refers to any cell, biological fluid or tissue. Suitable samples for use in this invention may include, without limitation, whole blood, leukocytes, fibroblasts, serum, urine, plasma, saliva, bone marrow, cerebrospinal fluid, amniotic fluid, and skin cells. Such samples may further be diluted with saline, buffer or a physiologically acceptable diluent. Alternatively, such samples are concentrated by conventional means.

[0058] “Patient” or “subject” as used herein means a male or female human, dogs, and animal models used for clinical research. In one embodiment, tire subject of these methods and compositions is a human diagnosed with CD2 -positive (CD20+) lymphoma in CNS, optionally wherein the CD20-positive lymphoma is CD20-positive B-cell Non-Hodgkins Lymphoma, a CD20-positive central nervous system (CNS) diffuse large B cell lymphoma (CNS-DLBCL), or CD20-positive B-cell secondary CNS lymphoma (SCNSL). In certain embodiments, the human subject of these methods and compositions is a prenatal, a newborn, an infant, a toddler, a preschool, a grade-schooler, a teen, a young adult or an adult.

[0059] With regard to the description of these various embodiments, it is intended that each of the compositions herein described, is useful, in another embodiment, in the methods of the invention. In addition, it is also intended that each of the compositions herein described as useful in the methods, is, in another embodiment, itself an embodiment of the invention.

[0060] Unless defined otherwise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application.

[0061] Nucleic Acid Molecules

[0062] In one aspect, a recombinant nucleic acid molecule comprising an engineered nucleic acid sequence which encodes an anti-hCD20 antibody (i.e., rituximab immunoglobulin) is provided (also referred to as rituximab-coGA, rituximab-coGS. rituximab-coGW. rituximab-coNP). In one aspect, an engineered nucleic acid sequence which encodes an anti-hCD20 antibody (i.e.. rituximab immunoglobulin) is provided (also referred to as rituximab-coGA, rituximab-coGS, rituximab-coGW, rituximab-coNP). Rituximab immunoglobulin comprises a heavy chain and a light chain. In one embodiment, the engineered nucleic acid sequence is useful to improve production, transcription, expression and / or safety in a subject. In another embodiment, the engineered sequence is useful to increase efficacy of the resulting therapeutic compositions or treatment. In a further embodiment, tire engineered sequence is useful to increase the efficacy of the rituximab immunoglobulin being expressed, but may also permit a lower dose of a therapeutic reagent that delivers tire immunoglobulin to increase safety. In certain embodiments, provided herein is a recombinant nucleic acid molecule comprising nucleic acid sequence encoding rituximab immunoglobulin. In certain embodiments, the recombinant nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 19, or a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, and including values therebetween, identical to SEQ ID NO: 19. In certain embodiments, the recombinant nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 19.

[0063] In certain embodiments, the recombinant nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 52, or a sequence at least 95%, at least 96%. at least 97%, at least 98%, at least 99%, at least 99.9%, and including values therebetween, identical to SEQ ID NO: 52. In certain embodiments, the recombinant nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 52. In certain embodiments, the recombinant nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 53, or a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, and including values therebetween, identical to SEQ ID NO: 53. In certain embodiments, the recombinant nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 53. In certain embodiments, the recombinant nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 54, or a sequence at least 95%. at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, and including values therebetween, identical to SEQ ID NO: 54. In certain embodiments, the recombinant nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 54.

[0064] In one embodiment, the engineered coding sequences for the heavy chain and light chain of rituximab are provided in SEQ ID NO: 19. In certain embodiments, the engineered coding sequences for the heavy chain and light chain of rituximab comprise nucleic acid sequence at least 95%, at least 96%. at least 97%, at least 98%, at least 99%. at least 99.9%, and including values therebetween, identical to SEQ ID NO: 19. In one embodiment, the engineered coding sequences for the heavy chain and light chain of rituximab are provided in SEQ ID NO: 52. In certain embodiments, the engineered coding sequences for tire heavy chain and light chain of rituximab comprise nucleic acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, and including values therebetween, identical to SEQ ID NO: 52. In one embodiment, the engineered coding sequences for tire heavy chain and light chain of rituximab are provided in SEQ ID NO: 53. In certain embodiments, the engineered coding sequences for the heavy chain and light chain of rituximab comprise nucleic acid sequence at least 95%, at least 96%. at least 97%, at least 98%, at least 99%. at least 99.9%, and including values therebetween, identical to SEQ ID NO: 53. In one embodiment, the engineered coding sequences for the heavy chain and light chain of rituximab are provided in SEQ ID NO: 54. In certain embodiments, the engineered coding sequences for the heavy chain and light chain of rituximab comprise nucleic acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, and including values therebetween, identical to SEQ ID NO: 54.

[0065] In certain embodiments, the engineered nucleic acid sequence encoding rituximab immunoglobulin comprises, 5’ to 3 ', an IL2 leader peptide, rituximab heavy chain, a furin cleavage sequence, optionally a “GSG” linker, a T2A linker, an IL2 leader peptide, optionally which is a modified IL2 leader peptide, and a rituximab light chain. In certain embodiment, the engineered nucleic acid sequence encoding rituximab immunoglobulin comprises, 5’ to 3’, an IL2 leader peptide, rituximab heavy chain, a furin cleavage sequence, “GSG” linker, a T2A linker, an IL2 leader peptide which is a modified IL2 leader peptide, and a rituximab light chain.

[0066] In certain embodiments, tire engineered nucleic acid sequence encoding rituximab immunoglobulin comprises nucleic acid sequence of SEQ ID NO: 19, wherein the sequence comprises sequences encoding an IL2 leader peptide sequence, a rituximab heavy chain, a furin / T2A linker (i.e.. furin cleavage site. “GSG” linker, and T2A linker), a modified 1L2 leader peptide sequence, and a rituximab light chain. In certain embodiments, the engineered nucleic acid sequence encoding rituximab immunoglobulin comprises nucleic acid sequence of SEQ ID NO: 52, wherein the nucleic acid sequence comprises sequences encoding an IL2 leader peptide sequence, a rituximab heavy chain, a furin / T2A linker, a modified IL2 leader peptide sequence, and a rituximab light chain. In certain embodiments, the engineered nucleic acid sequence encoding rituximab immunoglobulin comprises nucleic acid sequence of SEQ ID NO: 53, wherein tire nucleic acid sequence comprises sequences encoding an IL2 leader peptide sequence, a rituximab heavy chain, a furin / T2A linker, a modified IL2 leader peptide sequence, and a rituximab light chain. In certain embodiments, the engineered nucleic acid sequence encoding rituximab immunoglobulin comprises nucleic acid sequence of SEQ ID NO: 54, wherein tire nucleic acid sequence comprises sequences encoding an IL2 leader peptide sequence, a rituximab heavy chain, a furin / T2A linker, a modified IL2 leader peptide sequence, and a rituximab light chain.

[0067] In certain embodiments, provided herein is at least one open reading frame (ORF) comprising a nucleic acid sequence of SEQ ID NO: 20 encoding a leader peptide operably linked to rituximab heavy chain, a nucleic acid sequence comprising SEQ ID NO: 1 encoding a rituximab heavy chain, a nucleic acid sequence comprising SEQ ID NO: 37 encoding furin cleavage site, “GSG” linker and T2A linker, a nucleic acid sequence of SEQ ID NO: 22 encoding a leader peptide operably linked to a rituximab light chain, and a nucleic acid sequence comprising SEQ ID NO: 2 encoding a rituximab light chain (rituximab. coGA).

[0068] In certain embodiments, provided herein is at least one open reading frame (ORF) comprising a nucleic acid sequence of SEQ ID NO: 20 encoding a leader peptide operably linked to rituximab heavy chain, a nucleic acid sequence comprising SEQ ID NO: 3 encoding a rituximab heavy chain, a nucleic acid sequence comprising SEQ ID NO: 37 encoding furin cleavage site, '‘GSG” linker and T2A linker, a nucleic acid sequence of SEQ ID NO: 22 encoding a leader peptide operably linked to a rituximab light chain, and a nucleic acid sequence comprising SEQ ID NO: 4 encoding a rituximab light chain (rituximab. coGS).

[0069] In certain embodiments, provided herein is at least one open reading frame (ORF) comprising a nucleic acid sequence of SEQ ID NO: 20 encoding a leader peptide operably linked to rituximab heavy chain, a nucleic acid sequence comprising SEQ ID NO: 5 encoding a rituximab heavy chain, a nucleic acid sequence comprising SEQ ID NO: 37 encoding furin cleavage site. “GSG” linker and T2A linker, a nucleic acid sequence of SEQ ID NO: 22 encoding a leader peptide operably linked to a rituximab light chain, and a nucleic acid sequence comprising SEQ ID NO: 6 encoding a rituximab light chain (rituximab. coGW).

[0070] In certain embodiments, provided herein is at least one open reading frame (ORF) comprising a nucleic acid sequence of SEQ ID NO: 20 encoding a leader peptide operably linked to rituximab heavy chain, a nucleic acid sequence comprising SEQ ID NO: 7 encoding a rituximab heavy chain, a nucleic acid sequence comprising SEQ ID NO: 37 encoding furin cleavage site. “GSG” linker and T2A linker, a nucleic acid sequence of SEQ ID NO: 22 encoding a leader peptide operably linked to a rituximab light chain, and a nucleic acid sequence comprising SEQ ID NO: 8 encoding a rituximab light chain (rituximab. coNP).

[0071] A “nucleic acid”, as described herein, can be RNA, DNA, or a modification thereof, and can be single or double stranded, and can be selected, for example, from a group including nucleic acid encoding a protein of interest, oligonucleotides, nucleic acid analogues, for example peptide -nucleic acid (PNA), pseudocomplementary PNA (pc-PNA), locked nucleic acid (LNA) etc. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide (e.g., a peptide nucleic acid oligomer). The skilled man will appreciate that functional variants of these nucleic acid molecules are also intended to be a part of the present invention. Functional variants are nucleic acid sequences that can be directly translated, using the standard genetic code, to provide an amino acid sequence identical to that translated from the parental nucleic acid molecules. Such nucleic acid sequences include, for example, but are not limited to, In certain embodiments, tire recombinant nucleic acid molecule comprises expression cassette comprising at least one miRNA target sequences operably linked to a transgene, optionally in its 3’ UTR and / or its 5’ UTR. In certain embodiments, the recombinant nucleic acid molecule comprises a vector genome (comprising an expression cassette) which comprises at least one miRNA seed, binding site or full sequence. MicroRNAs (or miRNA or miR) are 19-25 nucleotide noncoding RNAs that bind to the sites of nucleic acid targets and down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation. In some embodiments, a microRNA sequence comprises a seed region, e.g., a sequence in the region of positions 2-8 of the mature microRNA, which has Watson-Crick sequence fully or partially complementarity to the miRNA target sequence of the nucleic acid. Such at least one miRNA may be used in combinations, including in an expression cassette or a vector genome also comprising a coding sequence for therapeutic protein, enzyme, or other moiety, and which is operably linked to the coding sequence. In certain embodiments, the vector genome may contain one miRNA to eight miRNA sequences, which are the same or different. Optionally, the vector genome does not contain therapeutic transgenes other than miRNA sequences.

[0072] In certain embodiments, tire miRNA is a dorsal root ganglion (drg)-spccific miRNA target sequence. In certain embodiments, the nucleic acid sequence further comprises at least one, at least two, at least three or preferably at least four tandem repeats of dorsal root ganglion (drg)- specific miRNA target sequences. In certain embodiments, the nucleic acid sequence further comprises four or more tandem repeats of dorsal root ganglion (drg)-specific miRNA target sequences. In certain embodiments, the nucleic acid sequence further comprises at least one. at least two, at least three, at least four, at least five, at least six, at least seven or preferably at least eight tandem repeats of dorsal root ganglion (drg)-specific miRNA target sequences. In some embodiments, the miRNA binding site is complementary to a miRNA expressed in a DRG (dorsal root ganglion) neuron, e.g., a miR183, and / or a miR182, binding site. In some embodiments, the miR binding site complementary to a miR expressed in expressed in a DRG neuron comprises a nucleotide sequence disclosed. See, e.g., PCT7US2019 / 67872, filed December 20, 2019, and now published as WO 2020 / 132455, which are incorporated herein by reference. See. also, International Patent Application No. PCT / US2021 / 32003, filed May 12, 2021, and now published WO2021 / 231579A1, which are incorporated herein by reference. See also, International Patent Application No. PCT / US2022 / 079881, filed November 22, 2022, and now published WO 2023 / 087019, which is incorporated herein by reference in its entirety.

[0073] In certain embodiments, tire miRNA is miR- 122 miRNA which modulates, e.g., reduces, the expression of a gene product in the liver.

[0074] In certain embodiments, provided herein are recombinant nucleic acid molecules encoding a rituximab immunoglobulin, and other constructs encompassed by the present invention and useful in generating expression cassettes and vector genomes which may be engineered for expression in yeast cells, insect cells or mammalian cells, such as human cells. Methods are known and have been described previously (e.g., WO 96 / 09378). A sequence is considered engineered if at least one non-preferred codon as compared to a wild type (WT) sequence is replaced by a codon that is more preferred. Herein, a non-preferred codon is a codon that is used less frequently in an organism than another codon coding for the same amino acid, and a codon that is more preferred is a codon that is used more frequently in an organism than a non-preferred codon. The frequency of codon usage for a specific organism can be found in codon frequency tables, such as in kazusa.jp / codon. Preferably more than one non-preferred codon, preferably most or all non-preferred codons, are replaced by codons that are more preferred. Preferably the most frequently used codons in an organism are used in an engineered sequence. Replacement by preferred codons generally leads to higher expression. It will also be understood by a skilled person that numerous different nucleic acid molecules can encode the same polypeptide as a result of the degeneracy of the genetic code. It is also understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the amino acid sequence encoded by the nucleic acid molecules to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed. Therefore, unless otherwise specified, a “nucleic acid sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleic acid sequences can be cloned using routine molecular biology techniques, or generated de novo by DNA synthesis, which can be performed using routine procedures by sendee companies having business in the field of DNA synthesis and / or molecular cloning (e.g., GeneArt, GenScript, Life Technologies. Eurofins).

[0075] In certain embodiments, the nucleic acid sequences encoding a rituximab immunoglobulin described herein are engineered (i.e.. assembled and placed) into any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, episome, etc., which transfers the rituximab immunoglobulin carried thereon to a host cell, e.g., for generating non-viral delivery systems (e.g., RNA-based systems, naked DNA, or the like), or for generating viral vectors in a packaging host cell, and / or for delivery to a host cells in a subject. In one embodiment, the genetic element is a vector. In one embodiment, the genetic element is a plasmid. The methods used to make such engineered constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g.. Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0076] It should be understood that the compositions in the rituximab immunoglobulin and rituximab immunoglobulin coding sequence described herein are intended to be applied to other compositions, regimens, aspects, embodiments and methods described across the Specification.

[0077] Expression Cassette

[0078] A gene therapy vector is provided herein which comprises an expression cassette comprising an engineered nucleic acid sequence comprising coding sequences for anti-hCD20 heavy chain and light chain, operably linked to regulatory sequences which direct expression thereof. Provided herein also is a recombinant nucleic acid molecule comprising expression cassette comprising nucleic acid sequence encoding for a rituximab immunoglobulin (i.e., anti- hCD20 heavy and light chain), as described herein. Provided herein also is a recombinant nucleic acid molecule comprising vector genome comprising nucleic acid sequence encoding for a rituximab immunoglobulin, as described herein. As used herein, an "expression cassette” refers to a nucleic acid molecule which comprises a biologically useful nucleic acid sequence (e.g., a gene cDNA encoding a protein, enzyme or other useful gene product, mRNA, etc.) and regulatory sequences operably linked thereto which direct or modulate transcription, translation, and / or expression of the nucleic acid sequence and its gene product. As used herein, “operably linked” sequences include both regulator}’ sequences that are contiguous or non-contiguous with the nucleic acid sequence and regulatory sequences that act in cis or trans with nucleic acid sequence. Such regulatory sequences typically include, e.g., one or more of a promoter, an enhancer, an intron, a Kozak sequence, a polyadenylation sequence, and a TATA signal. The expression cassette may contain regulatory sequences upstream (5’ to) of the gene sequence, e.g., one or more of a promoter, an enhancer, an intron, etc., and one or more of an enhancer, or regulatory sequences downstream (3’ to) a gene sequence, e.g., 3’ untranslated region (‘3 UTR) comprising a polyadenylation site, among other elements. In certain embodiments, the regulatory sequences are operably linked to the nucleic acid sequence of a gene product, wherein tire regulatory sequences are separated from nucleic acid sequence of a gene product by an intervening nucleic acid sequence(s), i.e., 5‘- untranslated regions (5 ’UTR). In some embodiments, the expression cassette can be a monocistronic or a bicistronic expression cassette.

[0079] In other embodiments, the term “transgene” refers to one or more DNA sequences from an exogenous source which are inserted into a target cell. Typically, an expression cassette contains the coding sequence for the gene product described herein flanked by packaging signals of the viral genome and other expression control sequences such as those described herein. In certain embodiments, a vector genome may contain two or more expression cassettes.

[0080] The term “exogenous” as used to describe a nucleic acid sequence or protein means that the nucleic acid or protein does not naturally occur in the position in which it exists in a chromosome, or host cell. An exogenous nucleic acid sequence also refers to a sequence derived from and inserted into the same host cell or subject, but which is present in a non-natural state, e.g., a different copy number, or under the control of different regulator}' elements.

[0081] The term “heterologous” as used to describe a nucleic acid sequence or protein means that the nucleic acid or protein was derived from a different organism or a different species of the same organism than the host cell or subject in which it is expressed. The term “heterologous” when used with reference to a protein or a nucleic acid in a plasmid, expression cassette, or vector, indicates that the protein or the nucleic acid is present with another sequence or subsequence which with which the protein or nucleic acid in question is not found in the same relationship to each other in nature.

[0082] As used herein, the term “regulatory sequence’’, or “regulatory' control sequences “or “expression control sequence” refers to nucleic acid sequences, such as initiator sequences, enhancer sequences, promoter sequences, intron sequences, and polyA signal sequences which direct, enable, induce, repress, or otherw ise control the transcription, translation and / or expression of protein encoding nucleic acid sequences to which they are operably linked.

[0083] In certain embodiments, the regulatory sequences comprise a promoter. In certain embodiment, the regulatory sequences comprise one or more intron(s), one or more enhancer(s). and a polyadenylation (polyA) signal sequence.

[0084] In one embodiment, the promoter is a ubiquitin C (UbC) promoter. See, e.g., WO 2001 / 091800. See, e.g., GenBank accession numbers AF232305 (rat) and D63791 (human), respectively. In certain embodiments, the UbC promoter comprises nucleic acid sequence of SEQ ID NO: 32.

[0085] However, other compositions containing the recombinant heavy chain or light chain sequences provided herein may utilize another selected promoters, e.g., a chicken P-actin (also referred to as chicken beta-actin, CB or CBA) promoter. A variety of chicken beta-actin promoters have been described alone, or in combination with various enhancer elements (e.g., CB7, also referred to as hybrid CB7) promoter comprising a cytomegalovirus immediate-early (CMV IE) enhancer and the chicken P-actin promoter, optionally with spacer sequence, optionally with a chimeric intron comprising chicken beta actin intron and further comprising a chicken beta-actin splicing donor (including the exon sequence, chicken beta actin intron) and rabbit beta-globin splicing acceptor), or a CBh promoter [SJ Gray et al, Hu Gene Ther, 201 1 Sep; 22(9): 143-1 153J. In certain embodiments, the promoter is cytomegalovirus (CMV) promoter. Still other promoters and / or enhancers may be selected. In another embodiment, a suitable promoter may include without limitation, an elongation factor 1 alpha (EF 1 alpha) promoter (see, e.g., Kim DW et al, Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system. Gene. 1990 Jul 16;91(2):217-23), a Synapsin 1 promoter (see, e.g., Kugler S et al, Human synapsin 1 gene promoter confers highly neuronspecific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. Gene Ther. 2003 Feb;10(4):337-47), a neuron- specific enolase (NSE) promoter (see, e.g., Kim J et al, Involvement of cholesterol-rich lipid rafts in interleukin-6- induced neuroendocrine differentiation of LNCaP prostate cancer cells. Endocrinology-. 2004 Feb;145(2):613-9. Epub 2003 Oct 16), or a CB6 promoter (see, e.g., Large-Scale Production of A deno- Associated Viral Vector Serotype-9 Carrying the Human Survival Motor Neuron Gene, Mol Biotechnol. 2016 Jan;58(l):30-6. doi: 10.1007 / s 12033-015-9899-5). In still other embodiments, multiple enhancers and / or promoters may be included.

[0086] In certain embodiments, an additional or alternative promoter sequence may be included as part of the expression control sequences (regulatory sequences), e.g., located between the selected enhancer or other regulatory sequence(s) and the coding sequence for the first antibody chain (e.g., the heavy chain). Constitutive promoters, regulatable promoters [see, e.g., WO 2011 / 126808 and WO 2013 / 04943], tissue specific promoters, or a promoter responsive to physiologic cues may be utilized in the vectors described herein. The promoter(s) can be selected from different sources, e.g., human cytomegalovirus immediate-early (CMV IE) enhancer / promoter, the SV40 early enhancer / promoter, the JC polymovirus promoter, myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoters, herpes simplex virus (HSV-1) latency associated promoter (LAP), rouse sarcoma virus (RSV) long terminal repeat (LTR) promoter, neuron-specific promoter (NSE), platelet derived growth factor (PDGF) promoter, hSYN, melanin-concentrating hormone (MCH) promoter, CBA, matrix metalloprotein promoter (MPP).

[0087] In addition to a promoter, an expression cassette may comprise one or more other appropriate transcription initiation sequences, transcription termination sequences, enhancer sequences, intron sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA for example WPRE; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.

[0088] In one embodiment, the regulatory' sequence further comprises an enhancer. In one embodiment, the regulatory sequence comprises one enhancer. In another embodiment, the regulatory- sequence contains two or more expression enhancers. These enhancers may be the same or may be different. For example, an enhancer may include an Alpha mic / bik enhancer or a cytomegalovirus immediate early (CMV IE) enhancer. This enhancer may be present in two copies which are located adjacent to one another. Alternatively, the dual copies of the enhancer may be separated by one or more sequences. In one embodiment, the regulatory- sequence further comprises an intron. In a further embodiment, the intron is a chicken beta-actin intron. In one embodiment, the intron is 875 bp (GenBank # X001 2. 1). In one embodiment, the intron is 973 bp intron from the chicken beta actin gene (GenBank # X00182. 1). In certain embodiments, the intron is a chimeric intron (CI) - a hybrid intron consisting of a human beta-globin splice donor and immunoglobulin G (IgG) splice acceptor elements. In certain embodiments, the intron is a chimeric intron comprising a chicken beta-actin splicing donor (including the exon sequence), chicken beta actin intron, and rabbit beta globin splicing acceptor. In another embodiment, a chimeric intron available from Promega® is used. In certain embodiments, the chimeric intron is a Promega® (chimeric) intron comprising nucleic acid sequence of SEQ ID NO: 33. Other suitable introns include those known in the art may by a human P-globulin intron, and / or a commercially available intron, and those described in WO 2011 / 126808.

[0089] In one embodiment, the regulatory sequence further comprises a polyadenylation signal (poly A). Examples of suitable polyA sequences include, e.g., rabbit beta globin (RBG or rBG) poly A, SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic polyAs. Optionally, one or more sequences may be selected to stabilize mRNA. In certain embodiments, the polyA is a rabbit beta globin poly A (rabbit globin polyA or rBG). See, e.g., WO 2014 / 151341. In certain embodiments, a human growth hormone (hGH) polyadenylation sequence, an SV40 polyA, or a synthetic polyA may be included in an expression cassette. In certain embodiments, the SV40 polyA is selected. In certain embodiments, the SV40 polyA comprises nucleic acid sequence of SEQ ID NO: 34.

[0090] In certain embodiments, the expression cassette comprises a rituximab coding sequence and may include other regulatory sequences therefor. The regulatory sequences necessary are operably linked to the rituximab coding sequence in a manner which permits its transcription, translation and / or expression in target cell.

[0091] In certain embodiment, the target cell may be a central nervous system cell. In certain embodiments, the target cell is one or more of an excitatory neuron, an inhibitor) neuron, a glial cell, a cortex cell, a frontal cortex cell, a cerebral cortex cell, a spinal cord cell. In certain embodiments, the target cell is in leptomeninges (LM) of the CNS. In certain embodiments, the target cell is in parenchyma of CNS.

[0092] In certain embodiments, the expression cassette comprises (i) a promoter optionally with enhancer, (ii) an intron, (iii) at least one ORF which comprises: a nucleic acid sequence encoding a leader peptide operably linked to a rituximab heavy chain, (iv) a nucleic acid sequence comprising SEQ ID NO: 1, 3, 5, or 7 or encoding a rituximab heavy chain, (v) optionally a furin cleavage site, (vi) optionally a “GSG” linker, (vii) a T2A element linker, (viii) a nucleic acid sequence encoding a leader peptide operably linked to a rituximab light chain, (ix) nucleic acid sequence comprising SEQ ID NO: 2, 4, 6 or 8 encoding a rituximab light chain, and (x) an SV40 polyadenylation (polyA) sequence.

[0093] In certain embodiments, the expression cassette comprises (i) a promoter which is a Ubiquitin C (UbC) promoter, (ii) an intron which is a chimeric intron comprising Promega intron (Pl), (iii) Kozak sequence, (iv) at least one ORF which comprises: a nucleic acid sequence encoding a leader peptide operably linked to a rituximab heavy chain, (v) a nucleic acid sequence comprising SEQ ID NO: 1, 3, 5 or 7 encoding a rituximab heavy chain, (vi) a furin cleavage site, (vii) a T2A element linker, (viii) a nucleic acid sequence encoding a leader peptide operably linked to a rituximab light chain, (ix) nucleic acid sequence comprising SEQ ID NO: 2, 4, 6 or 8 encoding a rituximab light chain, and (x) an SV40 polyadenylation (polyA) sequence.

[0094] In certain embodiments, the expression cassette comprises (i) a promoter which is a Ubiquitin C (UbC) promoter, (ii) an intron which is a chimeric intron comprising Promega intron (PI), (iii) Kozak sequence, (iv) at least one ORF which comprises: a nucleic acid sequence encoding a leader peptide operably linked to a rituximab heavy chain, (v) a nucleic acid sequence comprising SEQ ID NO: 1, 3, 5 or 7 encoding a rituximab heavy chain, (vi) a furin cleavage site, (vii) a “GSG” linker, (viii) a T2A element linker, (ix) a nucleic acid sequence encoding a leader peptide operably linked to a rituximab light chain, (x) nucleic acid sequence comprising SEQ ID NO: 2, 4, 6 or 8 encoding a rituximab light chain, and (xi) an SV40 polyadenylation (polyA) sequence.

[0095] In certain embodiments, the expression cassette comprises (i) a promoter which is a Ubiquitin C (UbC) promoter (SEQ ID NO: 32), (ii) an intron which is a chimeric intron comprising Promega intron (PI) (SEQ ID NO: 33), (iii) Kozak sequence, (iv) at least one ORF which comprises: a nucleic acid sequence encoding a leader peptide operably linked to a rituximab heavy chain (SEQ ID NO: 20), (v) a nucleic acid sequence comprising SEQ ID NO: 1, 3, 5 or 7 encoding a rituximab heavy chain, (vi) a furin cleavage site (SEQ ID NO: 35), (vii) a T2A element linker (SEQ ID NO: 36), (viii) a nucleic acid sequence encoding a leader peptide operably linked to a rituximab light chain (SEQ ID NO: 22), (ix) nucleic acid sequence comprising SEQ ID NO: 2, 4. 6 or 8 encoding a rituximab light chain, and (x) an SV40 polyadenylation (polyA) sequence (SEQ ID NO: 34).

[0096] In certain embodiments, the expression cassette comprises (i) a promoter which is a Ubiquitin C (UbC) promoter (SEQ ID NO: 32), (ii) an intron which is a chimeric intron comprising Promega intron (PI) (SEQ ID NO: 33), (iii) Kozak sequence, (iv) at least one ORF which comprises: a nucleic acid sequence encoding a leader peptide operably linked to a rituximab heavy chain (SEQ ID NO: 20), (v) a nucleic acid sequence comprising SEQ ID NO: 1, 3, 5 or 7 encoding a rituximab heavy chain, (vi) a furin cleavage site (SEQ ID NO: 35), (vii) “GSG” linker, (viii) a T2A element linker (SEQ ID NO: 36), (ix) a nucleic acid sequence encoding a leader peptide operably linked to a rituximab light chain (SEQ ID NO: 22), (x) nucleic acid sequence comprising SEQ ID NO: 2, 4, 6 or 8 encoding a rituximab light chain, and (xi) an SV40 polyadenylation (polyA) sequence (SEQ ID NO: 34).

[0097] In certain embodiments, tire expression cassette comprises (i) a promoter which is a Ubiquitin C (UbC) promoter (SEQ ID NO: 32), (ii) an intron which is a chimeric intron comprising Promega intron (PI) (SEQ ID NO: 33), (iii) Kozak sequence, (iv) at least one ORF which comprises: a nucleic acid sequence encoding a leader peptide operably linked to a rituximab heavy chain (SEQ ID NO: 20), (v) a nucleic acid sequence comprising SEQ ID NO: 5 encoding a rituximab heavy chain, (vi) a furin cleavage site (SEQ ID NO: 35), (vii) a T2A element linker (SEQ ID NO: 36), (viii) a nucleic acid sequence encoding a leader peptide operably linked to a rituximab light chain (SEQ ID NO: 22), (ix) nucleic acid sequence comprising SEQ ID NO: 6 encoding a rituximab light chain, and (x) an SV40 polyadenylation (polyA) sequence (SEQ ID NO: 34).

[0098] In certain embodiments, the expression cassette comprises (i) a promoter which is a Ubiquitin C (UbC) promoter (SEQ ID NO: 32). (ii) an intron which is a chimeric intron comprising Promega intron (PI) (SEQ ID NO: 33), (iii) Kozak sequence, (iv) at least one ORF which comprises: a nucleic acid sequence encoding a leader peptide operably linked to a rituximab heavy chain (SEQ ID NO: 20), (v) a nucleic acid sequence comprising SEQ ID NO: 5 encoding a rituximab heavy chain, (vi) a furin cleavage site (SEQ ID NO: 35), (vii) “GSG"’ linker, (viii) a T2A element linker (SEQ ID NO: 36), (ix) a nucleic acid sequence encoding a leader peptide operably linked to a rituximab light chain (SEQ ID NO: 22), (x) nucleic acid sequence comprising SEQ ID NO: 6 encoding a rituximab light chain, and (xi) an SV40 polyadenylation (polyA) sequence (SEQ ID NO: 34). In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 24 (UbC.PI.IL2_Vl.Rituximab-coGA) or a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99 to at least 100% identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 24 or a sequence at least 99% identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 24.

[0099] In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 26 (UbC.PI.IL2_Vl.Rituximab-coGS.SV40) or a sequence at least 95%, at least 96%, at least 97%, at least 98%. at least 99 to at least 100% identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 26 or a sequence at least 99% identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 26.

[0100] In certain embodiments, tire expression cassette comprises nucleic acid sequence of SEQ ID NO: 28 (UbC.PI.IL2_Vl.Rituximab-coGW.SV40) or a sequence at least 95%, at least 96%, at least 97%, at least 98%. at least 99 to at least 100% identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 28 or a sequence at least 99% identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 28.

[0101] In certain embodiments, tire expression cassette comprises nucleic acid sequence of SEQ ID NO: 30 (UbC.PI.IL2_Vl.Rituximab-coNP) or a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99 to at least 100% identical thereto. In certain embodiments, tire expression cassette comprises nucleic acid sequence of SEQ ID NO: 30 or a sequence at least 99% identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 30.

[0102] In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 65 (comprising rituximab. coGW) or a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99 to at least 100% identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 65 or a sequence at least 99% identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 65. It should be understood that the compositions in the expression cassette described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

[0103] Recombinant Adeno-associated Virus (rAAV)

[0104] Provided herein is a recombinant adeno-associated virus (rAAV) comprising an engineered nucleic acid sequence encoding rituximab immunoglobulin (e.g.. rAAV-anti-hCD20. rAAV-anti-hCD20-co, rAAV-Rituximab, rAAV.Rituximab-co, rAAV.Rituximab-coGA, rAAV.Rituximab-coGS, rAAV.Rituximab-coGW, rAAV.Rituximab-coNP). Provided herein is a recombinant adeno-associated virus (rAAV) useful for treating of CD20-positive CNS lymphoma. In certain embodiment, the rAAV comprises AAV capsid and an expression cassette. In certain embodiments, the rAAV comprises (a) an AAV capsid; and (b) a vector genome packaged in the AAV capsid of (a). Suitably, the AAV capsid selected targets tire cells to be treated. In certain embodiments, tire capsid is from Clade F. However, in certain embodiments, another AAV capsid source may be selected, i.e., Clade A. In certain embodiments, the AAV capsid is an AAVhu68 capsid. In certain embodiments, the AAV capsid is an AAVrh91 capsid. In certain embodiments, the AAV capsid is an AAVhu95 capsid. In certain embodiments, the AAV capsid is an AAV9 capsid. In certain embodiments, the AAV capsid is a mutant AAV9 capsid. In certain embodiments, tire AAV capsid is AAVhu96 capsid.

[0105] In certain embodiments, the provided herein is rAAV comprising a nucleic acid molecule comprising a vector genome comprising at least one AAV ITR at tire extreme 5' and / or extreme 3' end of the nucleic acid molecule which is the vector genome and an expression cassette. In certain embodiments, the vector genome is a nucleic acid molecule which comprises a 5' - AAV ITR, the expression cassette and a 3' - AAV ITR. In certain embodiments, wherein the vector genome comprises a nucleic acid sequence encoding a gene product operably linked to regulatory sequences which enables expression of transgene in a target cell, (e.g., by directing transcription, translation and / or expression).

[0106] In certain embodiments, tire vector genome comprises an AAV 5’ inverted terminal repeat (ITR), an expression cassette comprising rituximab engineered coding sequence comprising sequences encoding a rituximab heavy chain and a rituximab light chain and nucleic acid sequences operably linked thereto which regulate expression of the rituximab heavy and light chains, and an AAV 3’ ITR. In certain embodiments, the vector genome comprises an AAV 5’ inverted terminal repeat (ITR), an expression cassette comprising at least one open reading frame (ORF) comprising a rituximab heavy chain and a rituximab light chain and nucleic acid sequences operably linked thereto which regulate expression of the rituximab heavy and light chains, and an AAV 3 ’ ITR. In certain embodiments, , a vector genome contains, at a miniminn, from 5' to 3', an AAV 5’ ITR (also referred to as “AAV 5‘ ITR”, “5’ ITR”, “AAV 5' ITR”, or “5' ITR”), coding sequence(s) (e.g.,, transgene(s)), and an AAV 3’ ITR (also referred to as “AAV 3‘ ITR”. “3" ITR”, “AAV 3' ITR”, or “3' ITR”). In certain embodiments, , a vector genome contains, at a minimum, from 5' to 3', an AAV 5" ITR (also referred to as “AAV 5’ ITR”, “5?ITR”. “AAV 5' ITR”. or “5' ITR”), expression cassette comprising coding sequence(s), and an AAV 3’ ITR (also referred to as “AAV 3’ ITR”, “3’ ITR”, “AAV 3' ITR”, or “3' ITR”)

[0107] As used herein, the term “vector genome” refers to a nucleic acid molecule which is packaged in a viral capsid, for example, an AAV capsid, and is capable of being delivered to a host cell or a cell in a patient. In certain embodiments, the vector genome comprises terminal repeat sequences (e.g., AAV inverted terminal repeat sequences (ITRs) necessary for packaging the vector genome into the capsid at the extreme 5 ’ and 3 ‘ end and containing therebetween an expression cassette comprising the rituximab immunoglobulin gene (e.g., rituximab-coGA, rituximab-coGS, rituximab-coGW, rituximab-coNP) as described herein operably linked to sequences which direct expression thereof.

[0108] The AAV sequences of the vector typically comprise the cis-acting 5’ and 3’ inverted tenninal repeat (ITR) sequences (See, e.g., B. J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are about 145 base pairs (bp) in length. Preferably, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See. e.g., texts such as Sambrook et al. “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory. New York (1989); and K. Fisher et al., J. Virol., 70:520 532 (1996)). An example of such a molecule employed in the present invention is a “cis-acting” plasmid containing the transgene, in which the selected transgcnc sequence and associated regulatory elements arc flanked by the 5’ and 3’ AAV ITR sequences. In one embodiment, tire ITRs are from an AAV different than that supplying a capsid. In one embodiment, tire ITR sequences are from AAV2. A shortened version of the 5’ ITR, termed AITR, has been described in which the D-sequence and terminal resolution site (trs) are deleted. In certain embodiments, the vector genome (e.g., of a plasmid) includes a shortened AAV2 ITR of 130 base pairs, wherein the external A elements is deleted. The shortened ITR may revert back to the wild-type length of 145 base pairs during vector DNA amplification using the internal A element as a template and packaging into the capsid to form the viral particle. In other embodiments, the full-length AAV 5’ and 3’ ITRs are used. However, ITRs from other AAV sources may be selected. Where tire source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be termed pseudotyped. However, other configurations of these elements may be suitable.

[0109] In certain embodiments, provided herein is an rAAV comprising an AAV capsid and a nucleic acid molecule in the capsid. In certain embodiments, provided herein is an rAAV comprising an AAV capsid and a nucleic acid molecule comprising expression cassette. In certain embodiments, the nucleic acid molecule is a vector genome comprising at least one AAV ITR at the extreme 5' and / or extreme 3' end of the nucleic acid molecule which is the vector genome and an expression cassette. In certain embodiments, the vector genome is a nucleic acid molecule which comprises a 5' - AAV ITR (AAV 5' ITR), the expression cassette and a 3'- AAV ITR (AAV 3' ITR). In certain embodiments, the orientation of the ITRs may change from the orientation presented in the vector genome of the nucleic acid used in production (e.g.. a plasmid). In certain embodiments, tire rAAV may comprise a vector genome flanked by 3' and 5' AAV ITRs, respectively. In certain embodiments, the rAAV may comprise a vector genome flanked by two 5' AAV ITRs. In certain embodiments, the rAAV may comprise a vector genome flanked by two 3' AAV ITRs. In other embodiments, an rAAV as provided herein may be partially truncated such that the 5' AAV ITR and / or tire 3' AAV ITR is not detectable in the vector genome packaged in a final rAAV product.

[0110] In one embodiment. rAAV.rituximab vector has an AAV capsid and a vector genome packaged therein which comprises at least one element heterologous to AAV capsid. In one embodiment, rAAV.rituximab-coGA vector has an AAV capsid and a vector genome packaged therein which comprises at least one element heterologous to AAV capsid. In one embodiment, rAAV.rituximab-coGS vector has an AAV capsid and a vector genome packaged therein which comprises at least one clement heterologous to AAV capsid. In one embodiment, rAAV.rituximab-coGW vector has an AAV capsid and a vector genome packaged therein which comprises at least one element heterologous to AAV capsid. In one embodiment, rAAV.rituximab-coNP vector has an AAV capsid and a vector genome packaged therein which comprises at least one element heterologous to AAV capsid. In certain embodiments, the vector genome contains, from 5' to 3’: (a) an AAV ITR: (b) optionally an enhancer: (c) a promoter; (d) an intron; (e) a leader sequence and the rituximab heavy chain coding sequence; (f) optionally a furin cleavage sequence; (g) a T2A linker; (h) a leader sequence and the rituximab light chain coding sequence; (i) a polyA signal; and (j) an AAV ITR.

[0111] In certain embodiments, tire vector genome contains, from 5’ to 3’: (a) an AAV ITR; (b) optionally an enhancer; (c) a promoter; (d) an intron; (e) a leader sequence and the rituximab heavy chain coding sequence; (1) optionally a furin cleavage sequence; (g) optionally a “GSG linker”; (h) a T2A linker; (i) a leader sequence and the rituximab light chain coding sequence; (j) a polyA signal; and (k) an AAV ITR.

[0112] In certain embodiments, a rAAV may have AAV ITRs in an orientation of AAV 5' ITR - expression cassette - AAV 3' ITR. In certain embodiments, an rAAV has a vector genome in the orientation in which the 5' ITR and the 3' ITR have modified orientation in the final rAAV, as compared to the orientation of the vector genome in the production nucleic acid molecule (e.g., plasmid).

[0113] In one embodiment, the vector genome contains, from 5' to 3’: (a) an AAV ITR; (b) a Ubiquitin C (UbC) promoter; (c) a chimeric (Promega) intron (PI); (d) Kozak sequence; (e) a leader sequence and the rituximab heavy chain coding sequence: (f) a furin cleavage sequence; (g) a T2A linker; (h) a leader sequence and the rituximab light chain coding sequence; (i) an SV40 polyA signal; and (j) an AAV ITR.

[0114] In one embodiment, tire vector genome contains, from 5’ to 3’: (a) an AAV ITR; (b) a Ubiquitin C (UbC) promoter; (c) a chimeric (Promega) intron (PI); (d) Kozak sequence; (e) a leader sequence and the rituximab heavy chain coding sequence; (f) a furin cleavage sequence; (g) “GSG” linker: (j) a T2A linker; (i) a leader sequence and the rituximab light chain coding sequence; (j) an SV40 polyA signal: and (k) an AAV ITR

[0115] In one embodiment, the vector genome contains, from 5’ to 3’: (a) an AAV ITR; (b) a Ubiquitin C (UbC) promoter; (c) a chimeric (promega) intron (PI); (d) Kozak sequence; (e) a leader sequence and the rituximab heavy chain coding sequence; (f) a T2A linker; (g) a leader sequence and the rituximab light chain coding sequence; (h) an SV40 polyA signal; and (i) an AAV ITR.

[0116] In certain embodiments, the vector genome comprises nucleic acid sequence of SEQ ID NO: 25 (5’-ITR.UbC.PI.IL2_Vl.Rituximab-coGA.SV40.3’-ITR) or a sequence at least 95%, at least 96%, at least 97%. at least 98%, at least 99% to at least 100% identical thereto. In certain embodiments, tire vector genome comprises nucleic acid sequence of SEQ ID NO: 25 or a sequence at least 99% identical thereto. In certain embodiments, the vector genome comprises nucleic acid sequence of SEQ ID NO: 25.

[0117] In certain embodiments, tire vector genome comprises nucleic acid sequence of SEQ ID NO: 27 (5’-ITR. UbC.PI.IL2_Vl.Rituximab-coGS.SV40.3’-ITR) or a sequence at least 95%, at least 96%, at least 97%. at least 98%, at least 99% to at least 100% identical thereto. In certain embodiments, tire vector genome comprises nucleic acid sequence of SEQ ID NO: 27 or a sequence at least 99% identical thereto. In certain embodiments, the vector genome comprises nucleic acid sequence of SEQ ID NO: 27.

[0118] In certain embodiments, tire vector genome comprises nucleic acid sequence of SEQ ID NO: 29 (5’-ITR. UbC.PI.IL2_Vl.Rituximab-coGW.SV40.3’-ITR) or a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% to at least 100% identical thereto. In certain embodiments, tire vector genome comprises nucleic acid sequence of SEQ ID NO: 29 or a sequence at least 99% identical thereto. In certain embodiments, the vector genome comprises nucleic acid sequence of SEQ ID NO: 29.

[0119] In certain embodiments, the vector genome comprises nucleic acid sequence of SEQ ID NO: 31 (5’-ITR. UbC.PI.IL2_Vl.Rituximab-coNP.SV40.3’-ITR) or a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% to at least 100% identical thereto. In certain embodiments, the vector genome comprises nucleic acid sequence of SEQ ID NO: 31 or a sequence at least 99% identical thereto. In certain embodiments, the vector genome comprises nucleic acid sequence of SEQ ID NO: 31.

[0120] In certain embodiments, the vector genome comprises nucleic acid sequence of SEQ ID NO: 66 (comprising rituximab. coGW) or a sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% to at least 100% identical thereto. In certain embodiments, the vector genome comprises nucleic acid sequence of SEQ ID NO: 66 or a sequence at least 99% identical thereto. In certain embodiments, the vector genome comprises nucleic acid sequence of SEQ ID NO: 66.

[0121] In certain embodiments, the AAV capsid is Clade F AAV capsid, wherein the Clade F AAV capsid is selected from an AAVhu68 capsid [See. e.g., US2020 / 0056159;

[0122] PCI7US21 / 55436; SEQ ID NO: 38 and 39 for nucleic acid sequence; SEQ ID NO: 40 for amino acid sequence], an AAVhu95 capsid [See, e.g., International Patent Application No. PCT / US2022 / 077315, filed September 30, 2022; SEQ ID NOs: 46 and 47 (hu95 nucleic acid sequence)] and SEQ ID NO: 48 (hu95 amino acid sequence), or an AAVhu96 capsid [See, e.g., International Patent Application No. PCT / US2022 / 077315, filed September 30, 2022; SEQ ID NOs: 49 and 50 (hu96 nucleic acid sequence) and SEQ ID NO: 51 (hu96 amino acid sequence)], AAV9 capsid (SEQ ID NO: 41 for nucleic acid sequence; SEQ ID NO: 42 for amino acid sequence). In certain embodiments, the AAV capsid is a Clade A capsid, such as AAVrh91 capsid (nucleic acid sequence of SEQ ID NOs: 43 and 44; amino acid sequence of SEQ ID NO: 45). See, PCT / US20 / 030266, filed April 29. 2020, now published WO2020 / 223231, which is incorporated by reference herein and International Application No. PCT / US21 / 45945. filed August 13, 2021, which are incorporated herein by reference.

[0123] In certain embodiment, the AAV capsid is an AAVhu68 capsid. In certain embodiments the AAV capsid is an AAV9 capsid. In certain embodiments the AAV capsid is an AAVhu95 capsid. In certain embodiments, tire AAV capsid is an AAVhu96 capsid. In certain embodiments the AAV capsid is a mutant AAV9 capsid. See also, International Patent Application No. PCT / US2021 / 061312, filed December 1, 2021 (now published as WO 2022 / 119871 A2), and International Patent Application No. PCT / US2022 / 025879, filed April 22. 2022 (now published as WO 2022 / 226263 Al) which are all incorporated herein by reference in their entirety.

[0124] In certain embodiments, the AAV capsid for the compositions and methods described herein is chosen based on the target cell. In certain embodiment, the AAV capsid transduces a CNS cell and / or a PNS cell. In certain embodiments, other AAV capsid may be chosen, the AAV capsid is selected from a cy02 capsid, a rh43 capsid, an AAV8 capsid, a rhOl capsid, an AAV9 capsid, an rh8 capsid, a rhlO capsid, a bbOl capsid, a hu37 capsid, a rh02 capsid, a rh20 capsid, a rh39 capsid, a rh64 capsid, an AAV6 capsid, an AAV 1 capsid, a hu44 capsid, a hu48 capsid, a cy05 capsid a hul 1 capsid, a hu32 capsid, a pi2 capsid, or a variation thereof. In certain embodiments, the AAV capsid is a Clade F capsid, such as AAV9 capsid, AAVhu68 capsid, hu31 capsid, hu32 capsid, or a variation thereof. See, e.g., WO 2005 / 033321 published April 14, 2015, WO 2018 / 160582, US 2015 / 0079038, WO 2022 / 119871A2 published June 9, 2022, and WO 2022 / 226263A 1 published October 27, 2022, each of which is incorporated herein by reference in its entirety. In certain embodiments, the AAV capsid is a non-clade F capsid, for example a Clade A, B, C, D, or E capsid. In certain embodiment, the non-Clade F capsid is an AAV1 or a variation thereof. In certain embodiment, the AAV capsid transduces a target cell other than the nervous system cells. In certain embodiments, the AAV capsid is a Clade A capsid (e.g., AAV1, AAV6, AAVrh91). a Clade B capsid (e.g., AAV 2). a Clade C capsid (e.g., hu53). a Clade D capsid (e.g., AAV7), or a Clade E capsid (e.g., rhlO). In certain embodiments, the AAV capsid is an AAVrh32 / 33 capsid. See also, US 10,947,561 B2, which is incorporated herein by reference in its entirety .

[0125] An AAV capsid is an assembly of a heterogeneous population of vpl, a heterogeneous population of vp2, and a heterogeneous population of vp3 proteins. As used herein when used to refer to vp capsid proteins, the term “heterogeneous'’ or any grammatical variation thereof, refers to a population consisting of elements that are not the same, for example, having vpl, vp2 or vp3 monomers (proteins) with different modified amino acid sequences.

[0126] As used herein when used to refer to vp capsid proteins, the term “heterogeneous” or any grammatical variation thereof, refers to a population consisting of elements that are not the same, for example, having vpl, vp2 or vp3 (also referenced as VP1, VP2, VP3, or Vpl, Vp2, Vp3) monomers (proteins) with different modified amino acid sequences. The term “heterogeneous population” as used in connection with vpl, vp2 and vp3 proteins (alternatively termed isofomis), refers to differences in the amino acid sequence of the vpl, vp2 and vp3 proteins within a capsid. The AAV capsid contains subpopulations within the vpl proteins, within the vp2 proteins and within the vp3 proteins which have modifications from the predicted amino acid residues. These subpopulations include, at a minimum, certain deamidated asparagine (N or Asn) residues. For example, certain subpopulations comprise at least one, two, three or four highly deamidated asparagines (N) positions in asparagine - glycine pairs and optionally further comprising other deamidated amino acids, wherein tire deamidation results in an amino acid change and other optional modifications.

[0127] In certain embodiments. AAV capsids are provided which have a heterogeneous population of AAV capsid isoforms (i.e., VP1, VP2. VP3) which contain multiple highly deamidated “NG” positions. In certain embodiments, the highly deamidated positions are in the locations identified below, with reference to tire predicted full-length VP1 amino acid sequence. In other embodiments, the capsid gene is modified such that tire referenced “NG” is ablated and a mutant “NG” is engineered into another position. In certain embodiments, certain subpopulations comprise three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within tire capsid sequence. In certain embodiments, the VP1, VP2 VP3 proteins are a heterogenous population having deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, and / or N512, based on the AAV9 capsid residue positions. In certain embodiments, tire capsid comprises VP proteins which are highly deamidated in all of these positions. In certain embodiments, the percentage of deamidation in one or more of these highly deamidated positions is over 50%, over 55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, or about 70% to about 100%, or values therebetween.

[0128] As used herein, the terms ‘‘target cell’' and “target tissue” can refer to any cell or tissue which is intended to be transduced by the subject AAV vector. The term may refer to any one or more of muscle, liver, lung, airway epithelium, central nervous system, neurons, eye (ocular cells), or heart.

[0129] Additionally, provided herein is a recombinant nucleic acid molecule comprising (a) an AAV 5’ inverted terminal repeat (ITR), (b) an expression cassette (comprising at least one open reading frame (ORF)) comprising a rituximab heavy chain and a rituximab light chain and nucleic acid sequences operably linked thereto which regulate expression of the rituximab heavy chain and rituximab light chain, and (c) an AAV 3’ ITR, wherein tire expression cassette comprises: (i) a promoter which is a Ubiquitin C (UbC) promoter, and (ii) a intron, which is a chimeric intron (e.g., such as available from Promega), and (iii) at least one ORF which comprises: a nucleic acid sequence encoding a leader peptide operably linked to a rituximab heavy chain, (iv) a nucleic acid sequence comprising SEQ ID NO: 1, 3, 5 or 7 encoding a rituximab heavy chain, (v) a funn cleavage site, (vi) a T2A linker, (vii) a nucleic acid sequence encoding a leader peptide operably linked to a rituximab light chain, (viii) nucleic acid sequence comprising SEQ ID NO: 2, 4, 6 or 8 encoding a rituximab light chain, (ix) an SV40 polyadenylation (polyA) sequence, and wherein the expression cassette further comprises spacer sequences.

[0130] Additionally, provided herein is a recombinant nucleic acid molecule comprising (a) an AAV 5’ inverted terminal repeat (ITR), (b) an expression cassette (comprising at least one open reading frame (ORF)) comprising a rituximab heavy chain and a rituximab light chain and nucleic acid sequences operably linked thereto which regulate expression of the rituximab heavy chain and rituximab light chain, and (c) an AAV 3’ ITR, wherein the expression cassette comprises: (i) a promoter which is a Ubiquitin C (UbC) promoter, and (ii) a intron, which is a chimeric intron (e.g., such as available from Promega), and (iii) at least one ORF which comprises: a nucleic acid sequence encoding a leader peptide operably linked to a rituximab heavy chain, (iv) a nucleic acid sequence comprising SEQ ID NO: 1, 3, 5 or 7 encoding a rituximab heavy chain, (v) a furin cleavage site, (vi) “GSG linker”, (vii) a T2A linker, (viii) a nucleic acid sequence encoding a leader peptide operably linked to a rituximab light chain, (ix) nucleic acid sequence comprising SEQ ID NO: 2, 4, 6 or 8 encoding a rituximab light chain, (x) an SV40 poly adenylation (polyA) sequence, and wherein the expression cassette further comprises spacer sequences.

[0131] In certain embodiments, a recombinant nucleic acid molecule comprises expression cassette comprising nucleic acid sequence of SEQ ID NO: 24, 26, 28, 30 or 65. In certain embodiments, a recombinant nucleic acid molecule comprises vector genome comprising nucleic acid sequence of SEQ ID NO: 25. 27. 29, 31 or 66. In certain embodiments, a recombinant nucleic acid molecule is for use in rAAV production system as described herein.

[0132] Additionally, provided herein, is an rAAV production system useful for producing a rAAV as described herein. The production system comprises a cell culture comprising (a) a nucleic acid sequence encoding an AAV capsid protein; (b) the vector genome; and (c) sufficient AAV rep functions and helper functions to pennit packaging of the vector genome into the AAV capsid. In certain embodiments, the vector genome is SEQ ID NO: 25, T1. 29, 31 or 66. In certain embodiments, tire vector genome is SEQ ID NO: 29. In certain embodiments, the vector genome is SEQ ID NO: 66. In certain embodiments, the cell culture is bacterial cell culture. In certain embodiments, the cell culture is mammalian cell culture. In certain embodiments, the cell culture is a human embryonic kidney 293 (HEK293) cell culture. In certain embodiments, the cell culture is a suspension cell culture. In certain embodiments, tire AAV rep is from a different AAV. In certain embodiments, wherein the AAV rep is from AAV2. In certain embodiments, the AAV rep coding sequence and cap genes are on the same nucleic acid molecule, wherein there is optionally a spacer between the rep sequence and cap gene.

[0133] For use in producing an AAV viral vector (e.g., a recombinant (r) AAV), the vector genomes can be carried on any suitable vector, e g., a plasmid, which is delivered to a packaging host cell in culture and / or suspension. The plasmids useful in this invention may be engineered such that they are suitable for replication and packaging in vitro in prokary otic cells, insect cells, mammalian cells, among others. Suitable transfection techniques and packaging host cells arc known and / or can be readily designed by one of skill in the art.

[0134] In certain embodiments, a recombinant nucleic acid molecule is a plasmid. In certain embodiments, a recombinant nucleic acid molecule (e.g., plasmid) is useful in rAAV production. In certain embodiments, a recombinant nucleic acid molecule (e.g., a plasmid) useful in rAAV production comprises a vector genome comprising a 5?-ITR. UbC.PI.IL2 Vl.Rituximab- coGA.SV40.3’-ITR. In certain embodiments, recombinant a nucleic acid molecule (e.g., a plasmid) useful in rAAV production comprises a vector genome comprising a nucleic acid sequence of SEQ ID NO: 25. In certain embodiments, a recombinant nucleic acid molecule (e.g., a plasmid) useful in rAAV production comprises a vector genome comprising a 5’-ITR. UbC.PI.IL2_Vl.Rituximab-coGS.SV40.3’-ITR. In certain embodiments, recombinant a nucleic acid molecule (e.g., a plasmid) useful in rAAV production comprises a vector genome comprising a nucleic acid sequence of SEQ ID NO: 27. In certain embodiments, a recombinant nucleic acid molecule (e.g.. a plasmid) useful in rAAV production comprises a vector genome comprising a 5’-ITR. UbC.PI.IL2_Vl.Rituximab-coGW.SV40.3’-ITR. In certain embodiments, recombinant a nucleic acid molecule (e.g., a plasmid) useful in rAAV production comprises a vector genome comprising a nucleic acid sequence of SEQ ID NO: 29. In certain embodiments, recombinant a nucleic acid molecule (e.g., a plasmid) useful in rAAV production comprises a vector genome comprising a nucleic acid sequence of SEQ ID NO: 66. In certain embodiments, a recombinant nucleic acid molecule (e.g., a plasmid) useful in rAAV production comprises a vector genome comprising a 5’-ITR. UbC.PI.IL2 Vl.Rituximab-coNP.SV40.3’-ITR. In certain embodiments, recombinant a nucleic acid molecule (e.g., a plasmid) useful in rAAV production comprises a vector genome comprising a nucleic acid sequence of SEQ ID NO: 31.

[0135] Methods for generating and isolating AAVs suitable for use as vectors are known in the art. See generally, e.g., Grieger & Samulski, 2005, Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications, Adv. Biochem. Engin / Biotechnol. 99: 119-145; Buning et al., 2008, Recent developments in adeno-associated virus vector technology, J. Gene Med. 10:717-733; and the references cited below, each of which is incorporated herein by reference in its entirety. As used herein, a gene therapy vector refers to a rAAV as described herein, which is suitable for use in treating a patient. For packaging a gene into virions, the ITRs are the only AAV components required in cis in the same construct as the nucleic acid molecule containing the gene. The cap and rep genes can be supplied in trans. In one embodiment, tire expression cassettes described herein arc engineered into a genetic element (e.g., a shuttle plasmid) which transfers the immunoglobulin construct sequences carried thereon into a packaging host cell for production of a viral vector. In one embodiment, the selected genetic element may be delivered to an AAV packaging cell by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. Stable AAV packaging cells can also be made. The methods used to make such constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Molecular Cloning: A Laboratory Manual, ed. Green and Sambrook, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0136] The term ‘‘AAV intermediate” or “AAV vector intermediate” refers to an assembled rAAV capsid which lacks the desired genomic sequences packaged therein. These may also be termed an “empty” capsid. Such a capsid may contain no detectable genomic sequences of an expression cassette, or only partially packaged genomic sequences which are insufficient to achieve expression of the gene product. These empty capsids are non-functional to transfer the gene of interest to a host cell.

[0137] The recombinant adeno-associated virus (AAV) described herein may be generated using techniques which are known. See, e.g., WO 2003 / 042397; WO 2005 / 033321, WO 2006 / 110689; US 7588772 B2. Such a method involves culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; an expression cassette composed of. at a minimum, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the expression cassette into the AAV capsid protein. Methods of generating the capsid, coding sequences therefor, and methods for production of rAAV viral vectors have been described. See, e.g., Gao, et al, Proc. Natl. Acad. Sci. U.S.A. 100 (10), 6081-6086 (2003) and US 2013 / 0045186A1.

[0138] In certain embodiment, the rAAV are generated (manufactured) using triple transfection techniques. In certain embodiments the rAAV are generated using a stable mammalian cell line. In certain embodiments, the stable cell line comprises one or more of: (a) a first plurality of polynucleotide molecules which comprise a coding sequence for at least one adeno-associated virus (AAV) replicase (Rep) protein necessary for production of a replication-defective rAAV vector (Rep52 and Rep78), wherein said rep proteins coding sequences are operably linked to a doxycycline-inducible promoter which directs expression of the rep proteins in the cell line; (b) at least a second plurality’ of polynucleotide molecules each encoding adenovirus (Ad) helper proteins necessary for production of a replication-defective rAAV vector comprising at least an Ad E2A DNA Binding Protein (DBP) coding sequence, and Ad E4ORF6 coding sequence, wherein tire Ad E2A DBP coding sequences and the Ad E4ORF6 coding sequences are operably linked to a doxy cycline-inducible promoter which direct expression of the Ad helper proteins in the cell line; (c) a nucleic acid molecule comprising an Ad E 1 coding sequence operably linked to a constitutive promoter which directs expression of the Ad El in the cell line; (d) at least a third plurality of nucleic acid molecules each of which comprises an AAV VP1 coding sequence which encodes AAV VP 1 proteins, AAV VP2 proteins and AAV VP3 proteins which self-assemble to form an AAV capsid following expression in the cell, said AAV VP1 coding sequence being operably linked to a promoter which directs expression of the VP 1 coding sequences in the cell line.

[0139] In one embodiment, a production cell culture useful for producing a recombinant AAV having a capsid selected from an AAVhu68, an AAV9, a mutant AAV9. an AAVrh91. an AAVhu95 or an AAVhu96 is provided. Such a cell culture contains a nucleic acid which expresses the AAVhu68 capsid protein in the host cell (e.g., SEQ ID NO: 38 or SEQ ID NO: 39; a nucleic acid molecule suitable for packaging into the AAVhu68 capsid, e.g., a vector genome which contains AAV ITRs and a non-AAV nucleic acid sequence encoding a gene operably linked to regulatory sequences which direct expression of the gene in a host cell: and sufficient AAV rep functions and adenovirus helper functions to pennit packaging of the vector genome into tire recombinant AAVhu68, or AAVrh91 capsid (e.g., SEQ ID NO: 43 or SEQ ID NO: 44), AAVhu95 capsid (e.g., SEQ ID NO: 46 or SEQ ID NO: 47), AAVhu96 capsid (e.g., SEQ ID NO: 49 or SEQ ID NO: 50). In one embodiment, the cell culture is composed of mammalian cells (e.g., human embry onic kidney 293 cells, among others) or insect cells (e.g., Spodoptera frugiperda (Sf9) cells). In certain embodiments, baculovirus provides the helper functions necessary for packaging the vector genome into tire recombinant AAVhu68, AAVrh91, AAVhu95 or AAVhu96 capsid.

[0140] Optionally the rep functions are provided by an AAV other than AAV2, selected to complement the source of the ITRs.

[0141] In one embodiment, cells are manufactured in a suitable cell culture (e g., HEK 293 or Sf9) or suspension. Methods for manufacturing the gene therapy vectors described herein include methods well known in the art such as generation of plasmid DNA used for production of the gene therapy vectors, generation of the vectors, and purification of the vectors. In some embodiments, the gene therapy vector is an AAV vector and the plasmids generated are an AAV cis-plasmid encoding the AAV vector genome and the gene of interest, an AAV trans-plasmid containing AAV rep and cap genes, and an adenovirus helper plasmid. The vector generation process can include method steps such as initiation of cell culture, passage of cells, seeding of cells, transfection of cells with the plasmid DNA, post-transfection medium exchange to serum free medium, and the harvest of vector-containing cells and culture media. The harvested vectorcontaining cells and culture media are referred to herein as crude cell harvest. In yet another system, the gene therapy vectors are introduced into insect cells by infection with baculovirus- based vectors. For reviews on these production systems, see generally, e.g., Zhang et al., 2009, Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production, Human Gene Therapy 20:922-929, the contents of each of which is incorporated herein by reference in its entirety. Methods of making and using these and other AAV production systems are also described in the following U S patents, the contents of each of which is incorporated herein by reference in its entirety: US Patent Nos. 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065.

[0142] The crude cell harvest may thereafter be subject method steps such as concentration of the vector harvest, diafiltration of the vector harvest, microfluidization of tire vector harvest, nuclease digestion of the vector harvest, filtration of microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and / or formulation and filtration to prepare bulk vector. An affinity' chromatography purification followed anion exchange resin chromatography are used to purify the vector drug product and to remove empty capsids. These methods are described in more detail in International Patent Application No. PCT / US2016 / 065970, filed December 9, 2016, and US 11,098,286 B2, entitled “Scalable Purification Method for AAV9”, which are incorporated by reference. Purification methods for AAV8, International Patent Application No.

[0143] PCT / US2016 / 065976, filed December 9, 2016, and US 11,015,174 B2, entitled “Scalable Purification Method for AAV8”, which are incorporated herein by reference. Purification methods for rhlO, International Patent Application No. PCT / US16 / 066013, filed December 9, 2016, and US 11,028,372 B2, entitled “Scalable Purification Method for AAVrhlO”, which are incorporated herein by reference. Purification methods for AAV1, International Patent Application No. PCT / US2016 / 065974, filed December 9, 2016, and US 11,015,173 B2, entitled “Scalable Purification Method for AAV f’, which are incorporated herein by reference. Other suitable methods may be selected.

[0144] To calculate empty and full particle content. VP3 band volumes for a selected sample (e.g., in examples herein an iodixanol gradient-purified preparation where # of genome copies (GC) = # of particles) are ploted against GC particles loaded. The resulting linear equation (y = mx+c) is used to calculate tire number of particles in tire band volumes of the test article peaks. The number of particles (pt) per 20 ul. loaded is then multiplied by 50 to give particles (pt) / mb. Pt / mL divided by GC / mL gives the ratio of particles to genome copies (pt / GC). Pt / mL-GC / mL gives empty pt / mL. Empty pt / mL divided by pt / mL and x 100 gives the percentage of empty particles.

[0145] Generally, methods for assaying for empty capsids and AAV vector particles with packaged genomes have been known in the art. See. e.g., Grimm et al., Gene Therapy (1999) 6: 1322-1330: Sommer et al., Molec. Ther. (2003) 7: 122-128. To test for denatured capsid, the methods include subjecting the treated AAV stock to SDS-poly acrylamide gel electrophoresis, consisting of any gel capable of separating the three capsid proteins, for example, a gradient gel containing 3-8% Tris-acetate in the buffer, then running the gel until sample material is separated, and bloting the gel onto nylon or nitrocellulose membranes, preferably nylon. Anti-AAV capsid antibodies are then used as tire primary antibodies that bind to denatured capsid proteins, preferably an anti-AAV capsid monoclonal antibody, most preferably the Bl anti-AAV-2 monoclonal antibody (Wobus et al.. J. Virol. (2000) 74:9281-9293). A secondary antibody is then used, one that binds to the primary antibody and contains a means for detecting binding with the primary antibody, more preferably an anti-lgG antibody containing a detection molecule covalently bound to it, most preferably a sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase. A method for detecting binding is used to semi-quantitatively determine binding between the primary and secondary antibodies, preferably a detection method capable of detecting radioactive isotope emissions, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detection kit. For example, for SDS-PAGE. samples from column fractions can be taken and heated in SDS-PAGE loading buffer containing reducing agent (e.g., DTT), and capsid proteins were resolved on pre-cast gradient polyacrylamide gels (e.g.. Novex). Silver staining may be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions or other suitable staining method, i.e., SYPRO ruby or coomassie stains. In one embodiment, tire concentration of AAV vector genomes (vg) in column fractions can be measured by quantitative real time PCR (Q-PCR). Samples are diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After inactivation of the nuclease, the samples are further diluted and amplified using primers and a TaqMan™ Anorogenic probe specific for the DNA sequence between the primers. The number of cycles required to reach a defined level of fluorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism 7700 Sequence Detection System. Plasmid DNA containing identical sequences to that contained in the AAV vector is employed to generate a standard curve in the Q-PCR reaction. The cycle threshold (Ct) values obtained from the samples are used to determine vector genome titer by normalizing it to the Ct value of the plasmid standard curve. End-point assays based on the digital PCR can also be used.

[0146] In one aspect, an optimized q-PCR method is used which utilizes a broad spectrum serine protease, e.g.. proteinase K (such as is commercially available from Qiagen). More particularly, the optimized qPCR genome titer assay is similar to a standard assay, except that after the DN ase I digestion, samples are diluted with proteinase K buffer and treated with proteinase K followed by heat inactivation. Suitably samples are diluted with proteinase K buffer in an amount equal to the sample size. The proteinase K buffer may be concentrated to 2 -old or higher. Typically, proteinase K treatment is about 0.2 mg / mL, but may be varied from 0.1 mg / mL to about 1 mg / mL. The treatment step is generally conducted at about 55 °C for about 15 minutes, but may be performed at a lower temperature (e.g.. about 37 °C to about 50 °C) over a longer time period (e.g., about 20 minutes to about 30 minutes), or a higher temperature (e.g.. up to about 60 °C) for a shorter time period (e.g., about 5 to 10 minutes). Similarly, heat inactivation is generally at about 95 °C for about 15 minutes, but the temperature may be lowered (e g., about 70 to about 90 °C) and tire time extended (e.g., about 20 minutes to about 30 minutes). Samples are then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described in tire standard assay. Additionally, or alternatively, droplet digital PCR (ddPCR) may be used. For example, methods for determining single-stranded and self-complementary AAV vector genome titers by ddPCR have been described. See, e.g., M. Lock et al. Hu Gene Therapy Methods. Hum Gene Ther Methods. 2014 Apr;25(2): 115-25. doi: 10. 1089 / hgtb.2013. 131. Epub 2014 Feb 14.

[0147] In brief, the method for separating rAAVhu68 (or AAVrh91, AAVhu95 or AAVhu96) particles having packaged genomic sequences from genome-deficient AAVhu68 (or AAVrh91 or AAVhu95 or AAVhu96) intermediates involves subjecting a suspension comprising recombinant AAVhu68 (or AAVrh91) viral particles and AAVhu68 (or AAVrh91 or AVhu95 or AAVhu96) capsid intermediates to fast performance liquid chromatography, wherein the AAVhu68 (or AAVrh91 or AAVhu95 or AAVhu96) viral particles and AAVhu68 intermediates are bound to a strong anion exchange resin equilibrated at a pH of about 10.2 (or about 9.8 for AAVrh91), and subjected to a salt gradient while monitoring eluate for ultraviolet absorbance at about 260 nanometers (nm) and about 280 mn. Although less optimal for rAAVhu68 and AAVrh91. the pH may be in the range of about 10 to 10.4. In this method, the AAV full capsids are collected from a fraction which is eluted when the ratio of A260 / A280 reaches an inflection point. In one example, for the Affinity Chromatography step, the diafiltered product may be applied to an affinity^ resin (Life Technologies) that efficiently captures the AAV serotype. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins flow through the column, while AAV particles are efficiently captured.

[0148] The rAAV. Rituximab or r A AV. Rituximab-coX (e.g., rAAV.Rituximab-coGA (rAAV.UbC.Pl.IL2_Vl.Rituximab-coGA.SV40). rAAV.Rituximab-coGS (UbC.PI.IL2_V l.Rituximab-coGS), rAAV.Rituximab-coGW (UbC.PI.IL2_Vl.Rituximab- coGW), rAAV.Rituximab-coNP (UbC.PI.IL2_Vl.Rituximab-coNP.SV40)) is suspended in a suitable physiologically compatible composition (e.g., a buffered saline). This composition may be frozen for storage, later thawed and optionally diluted with a suitable diluent. Alternatively, the vector may be prepared as a composition which is suitable for delivery to a patient without proceeding through the freezing and thawing steps.

[0149] As used herein, the term ■’clade'’ as it relates to groups of AAV refers to a group of AAV 'hich are phylogenetically related to one another as determined using a Neighbor-Joining algorithm by a bootstrap value of at least 75% (of at least 1000 replicates) and a Poisson correction distance measurement of no more than 0.05, based on alignment of the AAV vpl amino acid sequence. The Neighbor- Joining algorithm has been described in the literature. See, e.g., M. Nei and S. Kumar, Molecular Evolution and Phylogenetics (Oxford University Press, New York (2000). Computer programs are available that can be used to implement this algorithm. For example, the MEGA v2.1 program implements the modified Nei-Gojobori method. Using these techniques and computer programs, and the sequence of an AAV vpl capsid protein, one of skill in the art can readily determine whether a selected AAV is contained in one of the clades identified herein, in another clade, or is outside these clades. See, e.g., G Gao, et al, J Virol, 2004 Jim; 78(10): 6381-6388, which identifies Clades A, B, C, D, E and F, and provides nucleic acid sequences of novel AAV, GcnBank Accession Numbers AY530553 to AY530629. See, also, WO 2005 / 033321, which is incorporated herein by reference.

[0150] Unless defined otherw ise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application.

[0151] Pharmaceutical Composition

[0152] In one aspect, provided herein is a pharmaceutical composition comprising a rAAV as described herein in a formulation buffer.

[0153] In one embodiment, the rAAV is formulated at about 1 x 109genome copies (GC) / mL to about 1 x 1014GC / mL. In a further embodiment, tire rAAV is formulated at about 3 x 109GC / mL to about 3 x 1013GC / mL. In yet a further embodiment, the rAAV is formulated at about 1 x 109GC / mL to about 1 x 1013GC / mL. In one embodiment, the rAAV is formulated at least 1 x 1011GC / mL.

[0154] Provided herein also is a composition comprising an rAAV as described herein and an aqueous suspension media. In certain embodiments, the suspension is formulated for intravenous deliver}’, intrathecal administration, or intracerebroventricular administration. In one aspect, the compositions contain at least one rAAV stock and an optional carrier, excipient and / or preservative.

[0155] As used herein, '‘carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in tire art. Supplementary active ingredients can also be incorporated into tire compositions. The phrase “pharmaceutically -acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host. Delivery vehicles such as liposomes, nanocapsules, nanoparticles, lipid nanoparticle (LNP), microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present invention into suitable host cells. In particular, the rAAV vector delivered vector genomes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or tire like.

[0156] In one embodiment, a composition includes a final formulation suitable for delivery to a subject, e.g., is an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration. Optionally, one or more surfactants are present in the formulation. In another embodiment, the composition may be transported as a concentrate which is diluted for administration to a subject. In other embodiments, the composition may be lyophilized and reconstituted at the time of administration.

[0157] A suitable surfactant, or combination of surfactants, may be selected from among nonionic surfactants that are nontoxic.

[0158] In one embodiment, the composition includes a carrier, diluent, excipient and / or adjuvant. Suitable carriers may be readily selected by one of skill in tire art in view of the indication for which tire transfer virus is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g.. phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The buffer / carrier should include a component that prevents the rAAV, from sticking to the infusion tubing but does not interfere with the rAAV binding activity in vivo. A suitable surfactant, or combination of surfactants, may be selected from among non-ionic surfactants that are nontoxic. In one embodiment, a difunctional block copolymer surfactant terminating in primary hydroxyl groups is selected, e.g., such as Poloxamer 188 (also known under the commercial names Pluronic® F68 [BASF], Lutrol® F68, Synperonic® F68, Kolliphor® P188) which has a neutral pH, has an average molecular weight of 8400. Other surfactants and other Poloxamers may be selected, i.e., nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide)) flanked by tw o hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (Macrogol-15 Hydroxystearate), LABRASOL (Polyoxy capryllic glyceride), polyoxy -oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are commonly named with the letter "P" (for poloxamer) followed by three digits: the first two digits x 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x 10 gives the percentage polyoxyethylene content. In one embodiment Poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005 % to about 0.001% of the suspension. In certain embodiments, the composition containing tire rAAV. Rituximab or r A AV. Rituximab. coX is delivered at a pH in the range of 6 to 8, or 7.2 to 7.8, or 7.5 to 8. For intrathecal delivery, a pH above 7.5 may be desired, e g., 7.5 to 8, or 7.8. For intravenous delivery, a pH of about 6.8 to about 7.2 may be desired.

[0159] In certain embodiments, the formulation may contain a buffered saline aqueous solution not comprising sodium bicarbonate. Such a formulation may contain a buffered saline aqueous solution comprising one or more of sodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride and mixtures thereof, in water, such as a Harvard’s buffer. In one embodiment, the buffer is PBS. In another embodiment, the buffer is an artificial cerebrospinal fluid (aCSF), e.g., Eliott’s formulation buffer; or Harvard apparatus perfusion fluid (an artificial CSF with final Ion Concentrations (in mM): Na 150; K 3.0; Ca 1.4; Mg 0.8; P 1.0; Cl 155). The aqueous solution may further contain Kolliphortg' P188, a poloxamer which is commercially available from BASF which was formerly sold under the trade name Lutrol® F68. The aqueous solution may have a pH of 7.2.

[0160] In another embodiment, the formulation may contain a buffered saline aqueous solution comprising 1 mM Sodium Phosphate (NasPOv), 150 mM sodium chloride (NaCl), 3mM potassium chloride (KC1), 1.4 mM calcium chloride (CaC12), 0.8 mM magnesium chloride (MgCh), and 0.001% poloxamer (e.g., Kolliphor®) 188, pH 7.2. See, e.g., harv ardapparatus.com / liarvard-apparatus-perfusion-fluid.html. In certain embodiments, Harv ard’s buffer is preferred due to better pH stability observed with Harvard’s buffer.

[0161] In certain embodiments, the formulation buffer is artificial CSF with Pluronic F68. In other embodiments, the formulation may contain one or more permeation enhancers. Examples of suitable permeation enhancers may include, e.g., mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprv late, sodium caprate, sodium lauryl sulfate, poly oxy ethyl ene-9-laurel ether, or EDTA.

[0162] Optionally, the compositions of the invention may contain, in addition to the rAAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0163] The compositions according to the present invention may comprise a pharmaceutically acceptable carrier, such as defined above. Suitably, the compositions described herein comprise an effective amount of one or more AAV suspended in a pharmaceutically suitable carrier and / or admixed with suitable excipients designed for delivery to the subject via injection, osmotic pump, intrathecal catheter, or for delivery by another device or route. In certain embodiments, an ommaya reservoir is used for delivery. In one example, the composition is formulated for intrathecal delivery. In one example, the composition is formulated for intravenous (iv) delivery. In one embodiment, a therapeutically effective amount of said vector is included in tire pharmaceutical composition. The selection of the carrier is not a limitation of the present invention. Other conventional pharmaceutically acceptable carrier, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0164] As used herein, the term "dosage" or “amount” can refer to the total dosage or amount delivered to the subject in the course of treatment, or tire dosage or amount delivered in a single unit (or multiple unit or split dosage) administration.

[0165] In certain embodiments, the compositions can be formulated in dosage units to contain an amount of rAAV that is in the range of about 1 x 109GC per gram of brain mass to about 1 x 1013genome copies (GC) per gram (g) of brain mass, including all integers or fractional amounts within the range and the endpoints. In another embodiment, the dosage is 1 x IO10GC per gram of brain mass to about 1 x IO13GC per gram of brain mass. In specific embodiments, tire dose of the vector administered to a patient is at least about 1.0 x 109GC / g, about 1.5 x 109GC / g, about 2.0 x 109GC / g. about 2.5 x 109GC / g. about 3.0 x 109GC / g, about 3.5 x 109GC / g, about 4.0 x 109GC / g, about 4.5 x 109GC / g, about 5.0 x 109GC / g, about 5.5 x 109GC / g, about 6.0 x 109GC / g, about 6.5 x 109GC / g, about 7.0 x 109GC / g, about 7.5 x 109GC / g, about 8.0 x 109GC / g, about 8.5 x 109GC / g, about 9.0 x 109GC / g, about 9.5 x 109GC / g, about 1.0 x IO10GC / g, about 1.5 x 1010GC / g, about 2.0 x 1010GC / g, about 2.5 x 1010GC / g, about 3.0 x 1010GC / g, about 3.5 x 1010GC / g, about 4.0 x 1010GC / g. about 4.5 x 1010GC / g, about 5.0 x 1010GC / g, about 5.5 x 1010GC / g, about 6.0 x IO10GC / g, about 6.5 x 1010GC / g, about 7.0 x IO10GC / g, about 7.5 x 1010

[0166] GC / g, about 8.0 x 1010GC / g, about 8.5 x 1010GC / g, about 9.0 x IO10GC / g, about 9.5 x 1010

[0167] GC / g, about 1.0 x 1011GC / g. about 1.5 x 1011GC / g, about 2.0 x 1011GC / g. about 2.5 x 1011

[0168] GC / g, about 3.0 x 1011GC / g, about 3.5 x 1011GC / g, about 4.0 x 1011GC / g, about 4.5 x 1011

[0169] GC / g, about 5.0 x 1011GC / g, about 5.5 x 1011GC / g, about 6.0 x 1011GC / g, about 6.5 x 1011

[0170] GC / g, about 7.0 x 1011GC / g, about 7.5 x 1011GC / g, about 8.0 x 1011GC / g, about 8.5 x 1011

[0171] GC / g, about 9.0 x 1011GC / g, about 9.5 x 1011GC / g, about 1.0 x 1012GC / g, about 1.5 x 1012

[0172] GC / g, about 2.0 x 1012GC / g, about 2.5 x 1012GC / g, about 3.0 x 1012GC / g, about 3.5 x 1012

[0173] GC / g, about 4.0 x 1012GC / g, about 4.5 x 1012GC / g, about 5.0 x 1012GC / g, about 5.5 x 1012

[0174] GC / g, about 6.0 x 1012GC / g, about 6.5 x 1012GC / g, about 7.0 x 1012GC / g, about 7.5 x 1012

[0175] GC / g, about 8.0 x 1012GC / g, about 8.5 x 1012GC / g, about 9.0 x 1012GC / g, about 9.5 x 1012 GC / g, about 1.0 x 1013GC / g, about 1.5 x 1013GC / g, about 2.0 x 1013GC / g, about 2.5 x 1013

[0176] GC / g, about 3.0 x 1013GC / g, about 3.5 x 1013GC / g, about 4.0 x 1013GC / g, about 4.5 x 1013

[0177] GC / g, about 5.0 x 1013GC / g, about 5.5 x 1013GC / g, about 6.0 x 1013GC / g, about 6.5 x 1013

[0178] GC / g, about 7.0 x 1013GC / g, about 7.5 x 1013GC / g, about 8.0 x 1013GC / g, about 8.5 x 1013

[0179] GC / g, about 9.0 x 1013GC / g, about 9.5 x 1013GC / g, or about 1.0 x 1014GC / g brain mass.

[0180] Also, the replication-defective virus compositions can be formulated in dosage units to contain an amount of replication-defective virus that is in the range of about 1.0 x 109GC to about 1.0 x 10lbGC (to treat an average subject of 70 kg in body weight) including all integers or fractional amounts within the range, and preferably 1.0 x 1012GC to 1.0 x 1014GC for a human patient. In one embodiment, the compositions are formulated to contain at least IxlO9, 2xl09, 3xl09, 4xl09, 5xl09, 6xl09, 7xl09, 8xl09, or 9xl09GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO10, 2xlO10, 3xl010, 4xlO10, 5xl010, 6xlO10, 7xlO10, 8xl010, or 9xlO10GC per dose including all integers or fractional amounts within the range. In another embodiment, tire compositions are formulated to contain at least IxlO11, 2x10”, 3X1011. 4X1011. 5x10”, 6x10”, 7xlOn, 8x10”, or 9xlOnGC per dose including all integers or fractional amounts within tire range. In another embodiment, the compositions are formulated to contain at least IxlO12, 2xl012, 3xl012, 4xl 012, 5xl 012, 6xl012, 7x l012, 8x l012, or 9xl012GC per dose including all integers or fractional amounts within the range. In another embodiment, tire compositions are formulated to contain at least IxlO13, 2xl013, 3xl013, 4xl013, 5xl013, 6xl013, 7xl013, 8xl013, or 9xl013GC per dose including all integers or fractional amounts within tire range. In another embodiment, the compositions are fonnulated to contain at least IxlO14, 2xl014, 3xl014, 4x1014. 5xl014, 6xl014, 7xl014, 8xl014, or 9xl014GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO15, 2xl015, 3xl015, 4xl015, 5xl015, 6xl015, 7xl015, 8xl015, or 9xl015GC per dose including all integers or fractional amounts within the range. In one embodiment, for human application the dose can range from IxlO10to about IxlO12GC per dose including all integers or fractional amounts within the range.

[0181] In certain embodiments, the composition is delivered intrathecally, optionally via intra- cistema magna (ICM) injection. In certain embodiments, the composition is delivered via intraparenchymal administration. In certain embodiments, the composition is delivered via Ommaya Reservoir delivery system. In one embodiment, the pharmaceutical composition comprising a rAAV as described herein is administrable at a dose of about 1 x 109GC per gram of brain mass to about 1 x 1013GO per gram of brain mass.

[0182] The rAAV, preferably suspended in a physiologically compatible carrier, may be administered to a human or non-human mammalian patient.

[0183] It should be understood that the compositions in the pharmaceutical composition described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

[0184] Uses and Regimens

[0185] In one aspect, a method is provided herein is a method of treating a human subject diagnosed with a CD20-positive lymphoma. In one aspect, a method is provided herein is a method of treating a human subject diagnosed with a CD20-positive central nervous system (CNS) lymphoma. In one aspect, a method is provided herein is a method of treating a human subject diagnosed with CD20-positive B-cell Non-Hodgkins Lymphoma, a CD20-positive central nervous system (CNS) diffuse large B cell lymphoma (CNS-DLBCL). CD20-positive B-cell secondary' CNS lymphoma (SCNSL), Burkitt lymphoma (BL), Burkitt-like lymphoma (BLL) or mature B-cell acute leukemia (B-AL). In certain embodiments, the method of treating CD20- positive CNS lymphoma comprises ameliorating one or more symptoms of CD20-positive CNS lymphoma in a subject.

[0186] In certain embodiments, a composition provided herein is useful for treating chronic lymphocytic leukemia (CLL). In certain embodiments, a composition provided herein is useful for treating one or more of: rheumatoid arthritis, granulomatosis with polyangiitis, microscopic polyangiitis, and pemphigus vulgaris. In certain embodiments, the composition may be coadministered in a regimen comprising a second active drug, e.g., a methotrexate. In still other embodiments, the rAAV compositions provided herein may be used in a regimen which further comprises co-administration with an immunosuppressive regimen.

[0187] A method provided herein comprises administering to a subject a suspension of an rAAV vector as described herein. In one embodiment, the method comprises administering to a subject having a CD20-positive CNS lymphoma, a suspension of a rAAV as described herein in a formulation buffer at a dose of about 1 x 109GC per gram of brain mass to about 1 x 1014GC per gram of brain mass. In another embodiment, the method comprises administering to a subject a suspension of a rAAV as described herein in a formulation buffer at a dose of about 1 x 109GC per kg of body weight to about 1 x 1014GC per kg of body weight. Suitable doses for systemic administration can be determined by one of skill in the art.

[0188] In one embodiment, the subject is delivered a therapeutically effective amount of the rAAV described herein. As used herein, a “therapeutically effective amount” refers to the amount of the composition comprising the nucleic acid sequence encoding rituximab immunoglobulin which delivers and expresses in the target cells an amount of immunoglobulin sufficient to achieve efficacy. The dosage is adjusted to balance the therapeutic benefit against any side effects and such dosages may vary depending upon the therapeutic application for which the recombinant vector is employed. The levels of expression of the transgene product can be monitored to determine the frequency of dosage resulting in viral vectors, preferably AAV vectors containing the transgene. Optionally, dosage regimens similar to those described for therapeutic purposes may be utilized for immunization using the compositions described herein.

[0189] Suitable, conventional, and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to a desired organ (e.g., brain, CSF, intranasal, intrathecal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, intraparenchymal, intracerebro ventricular, intrathecal, ICM, lumbar puncture and other parenteral routes of administration. Routes of administration may be combined, if desired.

[0190] Suitable volumes for delivery of these doses and concentrations may be determined by one of skill in tire art. For example, volumes of about 1 pL to 150 mL may be selected for CNS delivery, with tire higher volumes being selected for adults. Typically, for newborn infants a suitable volume is about 0.5 mL to about 10 mL, for older infants, about 0.5 mL to about 15 mL may be selected. For toddlers, a volume of about 0.5 mL to about 20 mL may be selected. For children, volumes of up to about 30 mL may be selected. For pre-teens and teens, volumes up to about 50 mL may be selected. In still other embodiments, a patient may receive an intrathecal administration in a volume of about 5 mL to about 15 mL are selected, or about 7.5 mL to about 10 mL. Other suitable volumes and dosages may be determined for CNS delivery, intratumoral delivery, and / or for systemic delivery (e.g., IV). The dosage will be adjusted to balance the therapeutic benefit against any side effects and such dosages may vary depending upon tire therapeutic application for which the recombinant vector is employed.

[0191] In one embodiment, the method comprises administering rAAV as described herein is to the subject in need. The above-described recombinant vectors may be administered or delivered to host or target cells according to published methods. The rAAV. preferably suspended in a physiologically compatible carrier, may be administered to a subject, human or non-human mammalian patient. In certain embodiments, for administration to a human patient, the rAAV is suitably suspended in an aqueous solution containing saline, a surfactant, and a physiologically compatible salt or mixture of salts. Suitably, the formulation is adjusted to a physiologically acceptable pH, e.g., in the range of pH 6 to 9, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8. As the pH of the cerebrospinal fluid is about 7.28 to about 7.32, for intrathecal delivery, a pH within this range may be desired; whereas for intravenous delivery, a pH of about 6.8 to about 7.2 may be desired. For intravenous delivery, a pH of about 6.8 to about 7.2 may be desired. However, other pHs within the broadest ranges and these subranges may be selected for other route of delivery.

[0192] Suitably, the pharmaceutical compositions, as described herein, and the uses thereof comprise delivering to the subject via injection, osmotic pump, intrathecal catheter, or for delivery by another device or route. In one example, tire composition is formulated for intrathecal delivery.

[0193] As used herein, the terms "‘intrathecal delivery” or “intrathecal administration” refer to a route of administration for drugs via an injection into the spinal canal, more specifically into the subarachnoid space so that it reaches the cerebrospinal fluid (CSF). Intrathecal delivery may include lumbar puncture, intraventricular (including intracerebroventricular (ICV)), suboccipital / intracistemal, and / or C 1-2 puncture. For example, material may be introduced for diffusion throughout the subarachnoid space by means of lumbar puncture. In another example, injection may be into the cistema magna. In certain embodiment, a rAAV, vector, or composition as described herein is administrated to a subject in need via the intrathecal administration. In certain embodiments, the intrathecal administration is performed as described in US Patent Publication No. 2018 / 0339065 Al, published November 29, 2019, which is incorporated herein by reference in its entirety. In certain embodiments, the CNS administration is performed using Ommaya Reservoir (also referred to as Ommaya device or Ommaya system).

[0194] As used herein, the terms “intracistcmal delivery” or “intracistcmal administration” (i.c., intra-cistema magna (ICM)) refer to a route of administration for drugs directly into the cerebrospinal fluid of the cistema magna cerebellomedularis, more specifically via a suboccipital puncture or by direct injection into the cistema magna or via permanently positioned tube. As used herein, the term “intraparenchymaF; "‘dentate nucleus" or IDN refers to a route of administration of a composition directly into dentate nuclei. IDN allows for targeting of dentate nuclei and / or cerebellum. In certain embodiments, the IDN administration is performed using ClearPoint® Neuro Navigation System (MRI Interventions, Inc., Memphis, TN) and ventricular cannula, which allows for MRI-guided visualization and administration. Alternatively, other devices and methods may be selected.

[0195] Suitably, tire pharmaceutical compositions, as described herein, and the uses thereof comprise delivering to tumor bed to the subject via surgical and non-surgical techniques. See also, Gilbert, A., and Machluf, M., Nano to micro delivery systems: targeting angiogenesis in brain tumors, J Angiogenes Res. 2010 Oct 8;2(l):20; Balyasnikova, IV., et al., Intranasal Delivery of Mesenchymal Stem Cells Significantly Extends Survival of Irradiated Mice with Experimental Brain Tumors, Molecular Therapy, 2014, 22( 1):P 140-P 148, epub January 1, 2014, which are incorporated herein by reference in its entirety. In certain embodiments, the pharmaceutical compositions, as described herein, and the uses thereof comprise directly delivering to the tumor or tumor bed in the CNS. In certain embodiments, the pharmaceutical compositions, as described herein, and the uses thereof comprise delivering via a systemic route.

[0196] In a further aspect, suitably, the compositions of the invention are designed so that rAAV vectors carry the nucleic acid expression cassettes encoding the rituximab immunoglobulin construct and regulatory sequences which direct expression of the rituximab immunoglobulin thereof in the selected cell. Following administration of the vectors into the CNS, the vectors deliver the expression cassettes to the CNS and express the proteinaceous immunoglobulin constructs in vivo.

[0197] A regimen as described herein may comprise, in addition to one or more of the combinations described herein, further combination with one or more of an anti-neoplastic biological drug, an anti-neoplastic small molecule drug, a chemotherapeutic agent, immune enhancers, radiation, surgery, and the like. A biological drug as described herein, is based on a peptide, polypeptide, protein, enzyme, nucleic acid molecule, vector (including viral vectors), or the like.

[0198] In certain embodiments, provided herein is a regimen comprising combination of rAAV, as described herein, and one or more chemotherapeutic regimens, including but not limited to cyclophosphamide, doxorubicin, vincristine, prednisone or other anthracycline-based chemotherapy regimens. In one embodiment, the method further comprises the subject receives an immunosuppressive co-therapy. Immunosuppressants for such co-therapy include, but are not limited to, a glucocorticoid, corticosteroids, antimetabolites, T-cell inhibitors, a macrolide (e.g., a rapamycin or rapalog), and cytostatic agents including an alkydating agent, an anti-metabolite, a cytotoxic antibiotic, an antibody, or an agent active on immunophilin. The immune suppressant may include a nitrogen mustard, nitrosourea, platinum compound, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, an anthracycline, mitomycin C, bleomycin, mithramycin, IL-2 receptor- (CD25-) or CD3-directed antibodies, anti-IL-2 antibodies, ciclosporin. tacrolimus, sirolimus. IFN-p. IFN-y, an opioid, or TNF-a (tumor necrosis factoralpha) binding agent.

[0199] In certain embodiments, tire immunosuppressive therapy may be started 0, 1, 2, 7, or more days prior to the gene therapy administration. Such therapy may involve co-administration of two or more drugs, the (e.g., prednelisone, micophenolate mofetil (MMF) and / or sirolimus (i.e., rapamycin)) on the same day. One or more of these drugs may be continued after gene therapy administration, at the same dose or an adjusted dose. Such therapy may be for about 1 week (7 days), about 60 days, or longer, as needed. In certain embodiments, a tacrolimus-free regimen is selected.

[0200] In one embodiment, the method further comprises administering to a subject anti-AAV neutralizing antibodies (NAb) to reduce peripheral transduction, and mitigate the potential risk of rituximab-induced toxicity. In certain embodiments, the method further comprises detect the presence of systemic AAV NAb prior to treating with anti-AAV NAb, wherein patients with levels of anti-AAV NAb in excess of a predetermined level against the rAAV capsid (or a sero- crossreactive capsid) do not require pretreatment. Such levels may be, e.g., in excess of about 1: 10, about 1 :20, about 1:50, about 1 : 100. about 1 :250, or higher or lower levels. In certain embodiments, the method further comprises intravenously administering human anti-AAV polyclonal antibodies (e.g., plasma-derived, pooled human immunoglobulin (IVIG)), an anti- AAV monoclonal antibody, or a cocktail of anti-AAV antibodies, to a patient about 1 day to about 2 hours before treatment with a rAAV-rituximab, e.g., rAAV.rituximab-coGA, rAAV.rituximab-coGS, rAAV.rituximab-coGW, or rAAV.rituximab-coNP. See also, WO 2023 / 196892A1 published October 12, 2023, which is incorporated herein by reference.

[0201] In certain embodiments, a combination regimen is provided for preventing off-target delivery rAAV, the regimen comprising (a) pretreating the patient by systemically administering a composition comprising anti-AAV capsid neutralizing antibodies directed against an AAV capsid in a recombinant AAV vector, and (b) administering to the central nervous system (CN S) rAAV as described herein (e.g., rAAV.rituximab). See also, International Patent Application No. PCT / US2023 / 065422, fded April 6, 2023, now Publication No. WO2023 / 196892A1 which is incorporated herein by reference in their entirety.

[0202] Still other co-therapeutics may include, e.g., anti-IgG enzymes, which have been described as being useful for depleting anti-AAV antibodies (and thus may permit administration to patients testing above a threshold level of antibody for the selected AAV capsid), and / or delivery of anti-FcRN antibodies and / or one or more of a) a steroid or combination of steroids and / or (b) an IgG-cleaving enzyme, (c) an inhibitor of Fc-IgE binding; (d) an inhibitor of Fc-IgM binding; (e) an inhibitor of Fc-IgA binding; and / or (f) gamma interferon. anti-FcRN antibodies include, e.g., rozanolixizumab (UCB7665) (UCB SA); IMVT-1401, RVT-1401 (HL161), HBM9161 (all form HanAll BioPhrma Co. Ltd), Nipocalimab (M281) (Momenta Pharmaceuticals Inc), ARGX-113 (efgartigimod) (Argenx S.E.), orilanolimab (ALXN 1830, SYNT001, Alexion Pharmaceuticals Inc), SYNT002, ABY-039 (Affibody AB), or DX-2507 (Takeda Pharmaceutical Co. Ltd). In certain embodiments, a combination of anti-FcRN antibodies is administered. In certain embodiments, an anti-FcRN antibody is administered in combination with a suitable anti- FcRn ligand (i.e., a peptide or protein construct binding human FcRn so as to inhibit IgG binding).

[0203] In one embodiment, a combination regimen for treating a patient with CD20-positive CNS lymphoma is provided, wherein the regiment includes administering a vector described herein in combination with a ligand which inhibits binding of human FcRn and pre-existing patient neutralizing antibodies (e.g.. IgG). In certain embodiments, the patient may be naive to any therapeutic treatment with a vector and may have pre-existing immunity due to prior infections with a wild-type virus. In other embodiments, the patient may have neutralizing antibodies as a result of a prior treatment or vaccination. In certain embodiments, the patient may have neutralizing antibodies 1: 1 to 1:20, or in excess of 1:2, in excess of 1:5, in excess of 1: 10, in excess of 1:20, in excess of 1:50, in excess of 1: 100, in excess of 1:200, in excess of 1:300 or higher. In certain embodiments, a patient has neutralizing antibodies in the range of 1 : 1 to 1 :200, or 1:5 to 1: 100, or 1:2 to 1: 20, or 1:5 to 1: 50, or 1:5 to 1:20. In certain embodiments, a patient receives a single anti-FcRn ligand (e.g.. anti-FcRn antibody) as the sole agent to modulate FcRn- IgG binding and to permit effective vector delivery. In other embodiments, a patient may receive a combination of one or more anti-FcRn ligands and a second component (e.g., an Fc receptor down-regulator (e.g., interferon gamma), an IgG enzyme, or another suitable component). Such combinations may be particularly desirable for patients having particularly high neutralizing antibody levels (e.g., in excess of 1:200).

[0204] In certain embodiments, an anti-FcRn ligand(s) (e.g., antibodies) is administered to a patient having neutralizing antibodies prior to and, optionally, concurrently with a selected viral vector. In certain embodiments, continued expression of an anti-FcRn ligand post administration of the gene therapy vector may desired on a short-term (transient basis), e.g., until such time as the viral vector clears from the patient. In certain embodiments, persistent expression of an anti- FcRn ligand may be desired. Optionally, in this embodiment, the ligand may be delivered via a viral vector, including, e.g., in the viral vector expressing the therapeutic transgene. However, this embodiment is not desirable where the therapeutic gene being delivered is an antibody or antibody construct or another construct comprising an IgG chain. In such embodiments, where an antibody construct having an IgG chain is being delivered via a viral vector to a patient having pre-existing immunity, the anti-FcRn ligand is delivered or dosed transiently so that the amount of anti-FcRn ligand in tire circulation is cleared from the sera before effective levels of vector- mediated transgene product are expressed.

[0205] In certain embodiments, the FcRn ligand is delivered one to seven days prior to administration of the vector (e.g., rAAV). In certain embodiments, the FcRn ligand is delivered daily. In certain embodiments, the FcRn ligand (e.g., immunoglobulin construct(s)) is delivered on the same day as the vector is administered. In certain embodiments, the FcRn ligand (e.g., immunoglobulin construct(s)) is delivered at least one day to four weeks post-rAAV administration. In certain embodiments, the ligand is delivered for four weeks to six months post- rAAV administration. In certain embodiments, the ligand is dosed via a different route of administration than the rAAV. In certain embodiments, the ligand is dosed orally, intravenously, or intraperitoneally. See also, International Patent Application No. PCT / US2021 / 037575, filed June 16, 2021, and now published WO 2021 / 257668 Al, which is incorporated herein by reference in its entirety.

[0206] In a combination therapy, the AAV -delivered immunoglobulin construct described herein is administered before, during, or after commencing therapy with another agent, as well as any combination thereof, i.e., before and during, before and after, during and after, or before, during and after commencing the anti-neoplastic therapy. For example, the AAV can be administered between 1 and 30 days, preferably 3 and 20 days, more preferably between 5 and 12 days before commencing radiation therapy. In another embodiment of the invention, chemotherapy is administered concurrently with or, more preferably, subsequent to AAV-mediated immunoglobulin (antibody) therapy. In still other embodiments, the compositions of the invention may be combined with other biologies, e.g., recombinant monoclonal antibody drugs, antibodydrug conjugates, or the like. Further, combinations of different AAV-delivered immunoglobulin constructs such as are discussed above may be used in such regimens.

[0207] Kit

[0208] In certain embodiments, a kit is provided which includes a concentrated vector suspended in a formulation (optionally frozen), optional dilution buffer, and devices and components required for intrathecal, intracerebroventricular or intracistemal administration. In another embodiment, the kit may additional or alternatively include components for intravenous delivery. In one embodiment, the kit provides sufficient buffer to allow for injection. Such buffer may allow for about a 1 : 1 to a 1 :5 dilution of the concentrated vector, or more. In other embodiments, higher or lower amounts of buffer or sterile water are included to allow for dose titration and other adjustments by the treating clinician. In still other embodiments, one or more components of the device are included in the kit. Suitable dilution buffer is available, such as, a saline, a phosphate buffered saline (PBS) or a glycerol / PBS.

[0209] It should be understood that tire compositions in kit described herein arc intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

[0210] Apparatus and Method for Delivery of a Pharmaceutical Composition

[0211] In one aspect, the vectors, rAAV or compositions thereof provided herein may be administered intrathecally via the method and / or the device provided in this section and described in WO 2017 / 136500 and WO 2018 / 160582, which are incorporated herein by reference. Alternatively, other devices and methods may be selected. In certain embodiments, tire method comprises the steps of CT-guided sub-occipital injection via spinal needle into the cistema magna of a patient. As used herein, the term Computed Tomography (CT) refers to radiography in which a three-dimensional image of a body structure is constructed by computer from a series of plane cross-sectional images made along an axis. In certain embodiments, the apparatus is described in US Patent Publication No. 2018-0339065 Al. published November 29, 2019, which is incorporated herein by reference in its entirety. In certain embodiments, the vectors, rAAV or compositions thereof provided herein may be administered using Ommaya Reservoir.

[0212] It should be understood that the compositions in the device described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

[0213] As used herein, the phrases "‘ameliorate a symptom”, “improve a symptom” or any grammatical variants thereof, refer to reversal of a CD20-positive CN S lymphoma-related symptoms. In one embodiment, the amelioration or improvement refers to the total number of symptoms in a patient after administration of the described composition(s) or use of the described method, which is reduced by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% compared to that before the administration or use. In another embodiment, the amelioration or improvement refers to the severity or progression of a symptom after administration of the described composition(s) or use of the described method, which is reduced by about 5%. about 10%. about 20%. about 30%. about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% compared to that before the administration or use.

[0214] As used herein, the “conservative amino acid replacement” or “conservative amino acid substitutions” refers to a change, replacement or substitution of an amino acid to a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity and size), which is known by practitioners of the art. Also see, e.g., FRENCH et al. What is a conservative substitution? Journal of Molecular Evolution, March 1983, Volume 19, Issue 2, pp 171-175 and YAMPOLSKY et al. The Exchangeability of Amino Acids in Proteins, Genetics. 2005 Aug: 170(4): 1459-1472, each of which is incorporated herein by reference in its entirety.

[0215] As used herein, the term “administration” or any grammatical variations thereof refers to delivery of composition described herein to a subject.

[0216] An “immunoglobulin molecule” is a protein containing the immunologically-activc portions of an immunoglobulin heavy chain and immunoglobulin light chain covalently coupled together and capable of specifically combining with antigen. Immunoglobulin molecules are of any type (e.g., IgG, IgE, IgM. IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4. IgAl and IgA2) or subclass. The terms "‘antibody’" and “immunoglobulin” may be used interchangeably herein.

[0217] As used herein, a “neutralizing antibody” or “NAb” binds specifically to a viral capsid or envelope and interferes with the infectivity of the virus or a recombinant viral vector having the viral capsid or envelope, thus preventing the recombinant viral vector from delivering effective amounts of a gene product encoded by an expression cassette in its vector genome. Various methods for assessing neutralizing antibodies in a patient's sera may be utilized. The term method and assay may be used interchangeably. As used herein, the term “neutralization assay” and “serum vims neutralization assay” refers to a serological test to detect the presence of systemic antibodies that may prevent infectivity of a vims. Such assays may also qualitatively or quantitatively discern the binding capacity (e.g., magnitude) or efficiency of the antibodies to neutralize a target. Immunological assays may include enzyme immunoassay (EIA), radioimmunoassay (RIA), which uses radioactive isotopes, fluoroimmunoassay (FIA) which uses fluorescent materials, chemiluminescent immunoassay (CLIA) which uses chemiluminescent materials and counting immunoassay (CIA) which employs particle-counting techniques, other modified assays such as western blot, immunohistochemistry (IHC) and agglutination. One of the most common enzyme immunoassays is enzyme-linked immunosorbent assay (ELISA).

[0218] “Neutralizing antibody titer” (NAb titer) a measurement of how much neutralizing antibody (e.g., anti-AAV NAb) is produced which neutralizes the physiologic effect of its targeted epitope (e.g., an AAV). Anti-AAV NAb titers may be measured as described in, e.g., Calcedo, R., et al., Worldwide Epidemiology of Neutralizing Antibodies to Adeno-Associated Viruses. Journal of Infectious Diseases, 2009, 199 (3): p. 381-390, which is incorporated by reference herein.

[0219] As used herein, a “subpopulation” of vp proteins refers to a group of vp proteins which has at least one defined characteristic in common and which consists of at least one group member to less than all members of the reference group, unless otherwise specified. For example, a “subpopulation” of vpl proteins is at least one (1) vpl protein and less than all vpl proteins in an assembled AAV capsid, unless otherw ise specified. A “subpopulation” of vp3 proteins may be one (1) vp3 protein to less than all vp3 proteins in an assembled AAV capsid, unless otherwise specified. For example, vpl proteins may be a subpopulation of vp proteins; vp2 proteins may be a separate subpopulation of vp proteins, and vp3 are yet a further subpopulation of vp proteins in an assembled AAV capsid. In another example, vpl, vp2 and vp3 proteins may contain subpopulations having different modifications, e.g., at least one, tw o. three or four highly deamidated asparagines, e.g., at asparagine - glycine pairs. Unless otherwise specified, highly deamidated refers to at least 45% deamidated, at least 50% deamidated, at least 60% deamidated, at least 65% deamidated, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 97%, 99%, up to about 100% deamidated, 50% to 100% deamidated, 70% to 100% deamidated, 75% to 100% deamidated, or 70% to 90% deamidated at a referenced amino acid position, as compared to the predicted amino acid sequence at the reference amino acid position. Such percentages may be determined using 2D-gel, mass spectrometry techniques, or other suitable techniques.

[0220] As used herein, a “stock” of rAAV refers to a population of rAAV. Despite heterogeneity in their capsid proteins due to deamidation, rAAV in a stock are expected to share an identical vector genome. A stock can include rAAV having capsids with, for example, heterogeneous deamidation patterns characteristic of the selected AAV capsid proteins and a selected production system. The stock may be produced from a single production system or pooled from multiple runs of the production system. A variety of production systems, including but not limited to those described herein, may be selected. See, e.g.. WO 2019 / 168961, published September 6, 2019, including Table G providing the deamidation pattern for AAV9 and WO 2020 / 160582, filed September 7, 2018. See, also, e.g., WO 2020 / 223231, published November 5, 2020 (rh91, including table with deamidation pattern), International Patent Application No. PCT / US21 / 45945, filed August 13, 2021, which are all incorporated herein by reference in its entirety.

[0221] The compositions described herein may be used in a regimen involving co-administration of other active agents. Any suitable method or route can be used to administer such other agents. Routes of administration include, for example, systemic, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration. Optionally, the AAV compositions described herein may also be administered by one of these routes.

[0222] The abbreviation “sc” refers to self-complementary. “Self-complementary AAV” refers a construct in which a coding region carried by a recombinant AAV nucleic acid sequence has been designed to form an intra-molecular double-stranded DNA template. Upon infection, rather than waiting for cell mediated synthesis of the second strand, the two complementary halves of scAAV will associate to form one double stranded DNA (dsDNA) unit that is ready for immediate replication and transcription. See, e.g., D M McCarty et al, “Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis”, Gene Therapy, (August 2001), Vol 8, Number 16, Pages 1248- 1254. Self-complementary A AVs are described in, e.g., U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety.

[0223] The term “heterologous” when used with reference to a protein or a nucleic acid indicates that tire protein or the nucleic acid comprises two or more sequences or subsequences which are not found in tire same relationship to each other in nature. For instance, tire nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid. For example, in one embodiment, the nucleic acid has a promoter from one gene arranged to direct the expression of a coding sequence from a different gene. Thus, with reference to the coding sequence, the promoter is heterologous.

[0224] A “replication-defective virus” or “viral vector” refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, where any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient; i.e., they cannot generate progeny virions but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not include genes encoding the enzymes required to replicate (the genome can be engineered to be "gutless" - containing only the transgene of interest flanked by the signals required for amplification and packaging of the artificial genome), but these genes may be supplied during production. Therefore, it is deemed safe for use in gene therapy since replication and infection by progeny virions camrot occur except in the presence of the viral enzyme required for replication.

[0225] A “recombinant AAV” or “rAAV” is a DNAse-resistant viral particle containing two elements, an AAV capsid and a vector genome containing at least non-AAV coding sequences packaged within the AAV capsid. In certain embodiments, the capsid contains about 60 proteins composed of vp 1 proteins, vp2 proteins, and vp3 proteins, which self-assemble to form the capsid. Unless otherwise specified, “recombinant AAV” or “rAAV” may be used interchangeably with the phrase “rAAV vector”. The rAAV is a “replication-defective virus" or "viral vector", as it lacks any functional AAV rep gene or functional AAV cap gene and cannot generate progeny. In certain embodiments, the only AAV sequences are the AAV inverted terminal repeat sequences (ITRs), typically located at the extreme 5’ and 3‘ ends of the vector genome in order to allow the gene and regulatory sequences located between the ITRs to be packaged within the AAV capsid. The term "‘nuclease-resistant” indicates that the AAV capsid has assembled around the expression cassette which is designed to deliver a transgene to a host cell and protects these packaged genomic sequences from degradation (digestion) during nuclease incubation steps designed to remove contaminating nucleic acids which may be present from the production process.

[0226] In many instances, rAAV particles are referred to as DNase resistant. However, in addition to this endonuclease (DNase), other endo- and exo- nucleases may also be used in the purification steps described herein, to remove contaminating nucleic acids. Such nucleases may be selected to degrade single stranded DNA and / or double-stranded DNA. and RNA. Such steps may contain a single nuclease, or mixtures of nucleases directed to different targets, and may be endonucleases or exonucleases.

[0227] As used herein, the term “host cell” may refer to the packaging cell line in which tire rAAV is produced from tire plasmid. In the alternative, the term “host cell” may refer to the target cell in which expression of tire transgene is desired.

[0228] As used herein, a “vector genome” refers to the nucleic acid sequence packaged inside the rAAV capsid which forms a viral particle. Such a nucleic acid sequence contains AAV inverted terminal repeat sequences (ITRs). In the examples herein, a vector genome contains, at a minimum, from 5’ to 3’, an AAV 5’ 1TR, expression cassette comprising coding sequence(s) (i.e., transgene(s)), and an AAV 3’ ITR. In certain embodiments, a vector genome comprises, at a minimum, from 5’ to 3’, an AAV ITR, expression cassette comprising coding sequence(s) (i.e., transgene(s)), and an AAV ITR. In certain embodiments, the ITRs are from AAV2, a different source AAV than the capsid, or other than full-length ITRs may be selected. In certain embodiments, the ITRs are from tire same AAV source as the AAV which provides the rep function during production or a trans-complementing AAV. Further, other ITRs. e.g., self- complementary (scAAV) ITRs, may be used. Both single -stranded AAV and self-complementary (sc) AAV are encompassed with the rAAV. The transgene is a nucleic acid coding sequence, heterologous to the vector sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest. The nucleic acid coding sequence is operatively linked to regulatory components in a manner which pennits transgene transcription, translation, and / or expression in a cell of a target tissue. Suitable components of a vector genome are discussed in more detail herein. In one example, a “vector genome” contains, at a minimum, from 5’ to 3’, a vector-specific sequence, a nucleic acid sequence encoding protein of interest operably linked to regulatory control sequences (which direct their expression in a target cell), where the vector-specific sequence may be a terminal repeat sequence which specifically packages the vector genome into a viral vector capsid or envelope protein. For example, AAV inverted terminal repeats are utilized for packaging into AAV and certain other parvovirus capsids.

[0229] In certain embodiments, non-viral genetic elements used in manufacture of a rAAV, will be referred to as vectors (e.g., production vectors). In certain embodiments, these vectors are plasmids, but the use of other suitable genetic elements is contemplated. Such production plasmids may encode sequences expressed during rAAV production, e.g., AAV capsid or rep proteins required for production of a rAAV, which are not packaged into the rAAV. Alternatively, such a production plasmid may carry the vector genome which is packaged into the rAAV.

[0230] As used herein, a ‘‘variant capsid” or a “variant AAV” or “variant AAV capsid” refers to a modified capsid, engineered capsid or a mutated capsid, wherein the capsid protein comprises an insertion of a tissue-specific targeting peptide, wherein modified insert is not a naturally occurring mutant.

[0231] The term “expression” is used herein in its broadest meaning and comprises the production of RNA or of RNA and protein. Expression may be transient or may be stable.

[0232] The term “substantial homology" or “substantial similarity,” when referring to a nucleic acid, or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95 to 99% of the aligned sequences. Preferably, the homology is over full-length sequence, or an open reading frame thereof, or another suitable fragment which is at least 15 nucleotides in length. Examples of suitable fragments are described herein.

[0233] The term “percent (%) identity”, “sequence identity”, “percent sequence identity”, or “percent identical” in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for correspondence. The length of sequence identity comparison may be over the full-length of the genome, the full-length of a gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides, is desired. However, identity among smaller fragments, e.g. of at least about nine nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be desired. Percent identity may be readily determined for amino acid sequences over the full-length of a protein, polypeptide, about 32 amino acids, about 330 amino acids, or a peptide fragment thereof or the corresponding nucleic acid sequence coding sequences. A suitable amino acid fragment may be at least about 7 amino acids in length, and may be up to about 700 amino acids. Examples of suitable fragments are described herein. By the term “highly conserved” is meant at least 80% identity, preferably at least 90% identity, and more preferably, over 97% identity’. Identity is readily determined by one of skill in tire art by resort to algorithms and computer programs known by those of skill in the art.

[0234] Generally, when referring to “identity”, “homology”, or “similarity” between two different sequences, “identity”, “homology” or “similarity” is determined in reference to “aligned” sequences. “Aligned” sequences or “alignments” refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence.

[0235] Identity may be determined by preparing an alignment of the sequences and through the use of a variety of algorithms and / or computer programs known in the art or commercially available (e.g., BLAST. ExPASy: Clustal Omega: FASTA; using, e.g., Needleman-Wunsch algorithm, Smith- Waterman algorithm). Alignments are performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs. Multiple sequence alignment programs are available for nucleic acid sequences. Examples of such programs include, “Clustal Omega”, “Clustal W”, “MUSCLE”, “CAP Sequence Assembly”, “BLAST”, “MAP”, and “MEME”, which are accessible through Web Servers on the internet. Other sources for such programs are known to those of skill in the art. Alternatively, Vector NTI utilities are also used. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using Fasta™, a program in GCG Version 10. 1. Fasta™ provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. For instance, percent sequence identity between nucleic acid sequences can be determined using Fasta™ with its default parameters (a word size of 6 and the NOPAM factor for tire scoring matrix) as provided in GCG Version 10. 1, herein incorporated by reference. Sequence alignment programs are also available for amino acid sequences, e g., the “Clustal Omega”. “Clustal X”, “MUSCLE”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “A comprehensive comparison of multiple sequence alignments”, 27(13):2682-2690 (1999).

[0236] In certain embodiments, an effective amount may be determined based on an animal model, rather than a human patient.

[0237] As described above, the term "‘about” when used to modify a numerical value means a variation of ±10%, (±10%, e.g.. ±1. ±2, ±3, ±4, ±5, ±6, ±7, ±8, ±9, ±10, or values therebetween) from the reference given, unless otherwise specified. In certain instances, the term “E±#” or the term “e±#” is used to reference an exponent. For example, “5E10” or “5el0” is 5 x 1010. These terms may be used interchangeably.

[0238] As used throughout this specification and the claims, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Herein, “up to” a number (for example, up to 50) includes tire number (for example, 50). The term “in the range” or “within a range” (and similar statements) includes the endpoints of the stated range.

[0239] As used throughout this specification and the claims, the terms “comprise” and “contain” and its variants including, “comprises”, “comprising”, “contains” and “containing”, among other variants, is inclusive of other components, elements, integers, steps and the like. The term “consists of’ or “consisting of’ are exclusive of other components, elements, integers, steps and the like.

[0240] It is to be noted that the term “a” or “an”, refers to one or more, for example, “an enhancer”, is understood to represent one or more enhancer(s). As such, the terms “a” (or “an”), “one or more,” and “at least one” is used interchangeably herein.

[0241] With regard to the description of these inventions, it is intended that each of the compositions herein described, is useful, in another embodiment, in the methods of the invention. In addition, it is also intended that each of tire compositions herein described as useful in the methods, is, in another embodiment, itself an embodiment of the invention.

[0242] Unless defined otherwise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application.

[0243] EXAMPLES

[0244] The following examples are provided to illustrate certain aspects of the claimed invention. The invention is not limited to these examples.

[0245] EXAMPLE 1 : Production of rAAV comprising engineered anti-CD20 antibody.

[0246] In the studies herein, various engineered sequences encoding anti-CD20 were generated, and comparative studies were performed. The rAAV are generated using triple transfection techniques, utilizing (1) a trans plasmid encoding AAV2 rep proteins and the AAVhu68 VP1 cap gene (alternatively AAVrh91. AAVhu95, AAV9, or a mutant AAV9). (2) a plasmid comprising adenovirus helper genes not provided by the packaging cell line which expresses adenovirus El a, and (3) a cis plasmid containing the vector genome for packaging in the AAV capsid. See, e.g., US 2020 / 0056159. The cis plasmid is designed to contain either the vector genome comprising engineered rituximab sequence (e.g., rituximab-coGA, rituximab-coGS, rituximab-coGW, rituximab-coNP) comprising IL-2 signal peptide and T2A element, optionally comprising a furin cleavage sequence, optionally with "GSG ' linker. The vector genomes include (among others): (i) (SEQ ID NO: 25); (ii) (SEQ ID NO: 27): (iii) (SEQ ID NO: 29); (iv) (SEQ ID NO: 31). or (v) (SEQ ID NO: 66). Additionally, the vector genomes include those comprising expression cassette including: (i) SEQ ID NO: 24, (ii) SEQ ID NO: 26, (iii) SEQ ID NO:28, (iv) SEQ ID NO: 30, or (v) SEQ ID NO: 65.

[0247] The vector genome contains an AAV 5’ inverted terminal repeat (ITR) and an AAV 3’ ITR at the extreme 5’ and 3’ end, respectively. The ITRs flank the sequences of the expression cassette packaged into the AAV capsid which have sequence encoding a rituximab. The expression cassette further comprises regulatory sequences operably linked to the fusion protein coding sequences, including a promoter (Ubiquitin C (UbC) promoter), an intron (chimeric intron comprising a Promega intron), SV40 polyA.

[0248] EXAMPLE 2: Evaluation of rAAV encoding ant-CD20 antibody.

[0249] Central nervous system lymphoma (CNSL) is a group of rare incurable malignancies that originate in the brain or spinal cord (primary CNSL) or from systemic disease that metastasizes to the CNS (secondary- CNSL). Up to 95% of CNSL cases arise from B-cell non-Hodgkin lymphoma (NHL). Although there is a lack of consensus, the 5-year survival rate for patients diagnosed with CNSL is <30%, and the overall survival from diagnosis is estimated to be <7 months for patients aged 70 years or older. Rituximab, a human / murine chimeric anti-CD20 monoclonal antibody, is part of the standard of care regimens for the treatment of indolent and aggressive forms of B-cell NHL. However, rituximab has low bioavailability- in the CNS because of the intrinsic properties of the blood-brain barrier.

[0250] In the studies below, we establish a model for rituximab expression mediated by an recombinant adeno-associated virus (rAAV) vector (rAAV.Rituximab or AAV.Rituximab) administered directly to the CN S delivery to bypass the blood-brain barrier and maximize the effectiveness of rituximab against B-cell CNSL. The aim is to evaluate transgene expression and antitumor efficacy of AAV.Rituximab against xenograft mouse models of CD2- positive (CD20+) CNSL following intracerebroventricular (ICV) vector delivery. This gene therapy approach may provide long term local delivery of rituximab to treat CD20-positive metastatic brain lesions following a single administration.

[0251] AAV vector technology allows for sufficient expression of anti-hCD20 antibody (rituximab) within the central nervous system (CNS). The expression of anti-hCD20 from an AAV vector would provide an effective one-time gene therapy approach for the treatment of CD20-positve primary and secondary CNS lymphomas. The current treatment strategies to manage CNS diffuse large B cell lymph (CNS-DLBCL) and secondary- CNS lymphomas (SCNSL) uses with a combination of non-surgical approaches comprising corticosteroids, systemic and intrathecal chemotherapy, and whole brain radiation therapy. Intracranial delivery of AAV.anti-hCD20 (also referred to as AAV.rituximab) could also significantly delay the emergence of new tumor lesions. CNS delivery of AAV.rituximab alone or in combination with standard-of-care therapies could be used as an alternative approach to achieve disease remission or stabilization since it could avoid treatment scape and result in long lasting antibody expression.

[0252] To achieve sustained CNS expression of rituximab, we developed a gene therapyapproach to treat CD20-positivc CNS lymphoma comprising a single local administration of a recombinant adeno-associated viral (rAAV) vector encoding for anti-hCD20 immunoglobulin (rituximab). To evaluate rituximab expression efficiency within the CNS, we engineered 4 new AAV.rituximab vectors: AAV.rituximab.coGA (AAVhu68.UbC.PI.IL2 Vl.Rituximab- coGA.SV40), AAV.rituximab.coGS (AAVhu68.UbC.PLIL2_VLRituximab-coGS.SV40), A AV. rituximab. coGW (AAVhu68.UbC.PI.IL2 Vl.Rituximab-coNP.SV40), and

[0253] A AV. rituximab. coNP (AAVhu68.UbC.PI.IL2_Vl.Rituximab-coGW.SV40), comprising expression cassette comprising a coding sequence for an engineered anti-hCD20 heavy chain and anti-hCD20 light chain under the transcriptional control of human ubiquitin C promoter (UbC), wherein the expression cassette further comprises a native interleukin-2 (IL2) signal peptide sequence preceding the antibody heavy chain sequence, a heavy chain DNA sequence, a furin site, "GSG” linker, T2A self-cleavage site, an IL2 signal peptide variant 1, and light chain DNA sequence. All rAAV vector iterations were packaged into AAVhu68 capsid and injected via the intracerebroventricular (1CV) route in 7-8-week-old adult Ragl knockout (KO) mice of both sexes (N= 5 mice / vector group) for subsequent gene expression studies. Phosphate buffered saline (PBS) injected Ragl KO mice were used as vehicle sham control group (N= 2 mice). The Ragl KO mouse model was chosen for this purpose since it is the preferred model for tumor challenge studies to confirm CD20 targeting using human-derived xenograft tumor models.

[0254] To quantify rituximab expression, we conducted a 28-day time-course ELISA (enzyme linked immunosorbent assay) analysis study using serum and brain homogenates (at endpoint) from whole-body -perfused mice treated ICV with 1x1011(1E11) genome copies (GC) / mouse of each A A Vhu68. rituximab vectors or PBS. We assessed vector biodistribution within the brain and liver to help determine the extent of brain and peripheral transduction following ICV administration. All vectors tested were well tolerated in mice and raised no safety concerns.

[0255] FIG. 2A shows biodistribution analysis with plotted measurement of DNA concentration (GC / pg) in tire collected liver and brain tissue samples following administration with AAV.rituximab.coGA, AAV. rituximab. coGS. AAV. rituximab. coGW. and AAV. rituximab. coNP. FIG. 2B shows biodistribution analysis with plotted measurement of RNA concentration (total RNA copies / lOOng) in the collected liver and brain tissue samples following administration with AAV.rituximab.coGA, AAV.rituximab.coGS, AAV.rituximab.coGW, and AAV.rituximab.coNP. FIG. 2C shows results of the ELISA with plotted measurements of rituximab concentration (ng / mg protein) in the perfused brain tissue samples collected post administration with AAV.rituximab.coGA, AAV.rituximab.coGS, AAV.rituximab.coGW, and AAV.rituximab.coNP. FIG. 2D show s results of the ELISA with plotted measurements of rituximab concentration (pg / mL) in the serum samples collected at day 0, 7, 14, and 30 post administration with AAV.rituximab.coGA, AAV.rituximab.coGS. AAV.rituximab.coGW. and AAV.rituximab.coNP. FIG. 3 A shows a representative western blot confirming expression of the heavy and light chain of rituximab in brain tissue following administration with AAV.rituximab.coGW. FIG. 3B shows a plot of the relative protein level of the heavy and light chain rituximab expression in brain tissue following administration with AAV.rituximab.coGW. Rituximab was detected by ELISA in the serum and brain of mice following single ICV delivery of each of all 4 vectors. Construct encoding for a rituximab. coGW sequence (AAVhu68.UbC.CI.IL2_Vl.Rituximab-coGW.SV40) resulted in highest expression in tire brain 28 days after ICV delivery (310 ng of antibody per mg of protein, vs. rituximab. coGA: 136 ng / mg; rituximab. coGS: 123.4 ng / mg; rituximab. coNP: 100.6 ng / mg ). To confirm rituximab identity, saline-perfused brain homogenates from a mouse that received ICV AAV. Rituximab was subjected to a rituximab pull-down enrichment assay using CD20-coated beads prior to liquid chromatography-mass spectrometry (LC-MS) using unique signature try ptic peptides indicating rituximab identity' (data not shown). Relative post-translational modification quantities between neat Rituximab control and AAVhu68. Rituximab dosed mouse are similar. Table A. To account for patients with pre-existing levels of anti-AAV antibodies in future clinical trials, which reduces systemic but has minimal to no impact on CNS transduction, we investigated the use of plasma-derived, pooled human immunoglobulin (IVIG) as a source of anti-AAV neutralizing antibodies to reduce peripheral transduction resulting from vector leakage into the systemic circulation. Compared to the control group that received intravenous (i.v.) naive mouse serum, Ragl KO mice administered IVIG intravenously (i.v.) two hours prior to ICV injection of AAVhu68.UbC.CI.IL2_Vl.Rituximab-coGW.SV40 vector exhibited only 2.8-fold lower levels of rituximab within the brain, albeit this difference didn't reach statistical significance (p=0.055). However, effective blockade of peripheral transduction resulted in 100- fold reduction in serum levels of rituximab (p=0.007). These results suggest presence of preexisting neutralizing antibodies against AAV prior should not preclude a patient from receiving the vector. FIG. 1A shows rituximab expression in serum in Ragl KO mice treated with ICV with 1 x 1011GC of AAVhu68.Rituximab.coGW with and without IVIG, and in Dulbecco's phosphate -buffered saline (DPBS) treated control group. FIG. IB shows rituximab expression in perfused brain in Ragl KO mice treated with ICV with 1 x IO11GC of AAVhu68.Rituximab.coGW with and without IVIG, and in Dulbecco's phosphate-buffered saline (DPBS) treated control group. FIG. 1C shows levels of rituximab DNA, plotted as GC / diploid cell, as measured in brain and liver tissue in Ragl KO mice treated with ICV with 1 x IO11GC of AAVhu68.Rituximab.coGW with and without IVIG. FIG. ID shows levels of rituximab RNA, plotted as GC / diploid cell, as measured in brain and liver tissue in Ragl KO mice treated with ICV with 1 x IO11GC of AAVhu68.Rituximab.coGW with and without IVIG.

[0256] ICV AAV. Rituximab Administration Delayed Tumor Progression in Cell Line- Derived Xenograft Mouse Models Of CNSL

[0257] In these studies, 6-8 week-old Ragl KO mice (males and females) were implanted with guide screw into the brain at day -21 to -7 of the study. On day 0, mice were implanted with Ramos , Daudi , or Oci-ly3 human lymphoma cell lines in the brain parenchyma using the guidescrew method. Three to 4 days following tumor implantation, mice received 1x1011GC of AAV. Rituximab (AAVhu68.Rituximab.coGW) or AAV.isotype control via ICV injection. The study was continued until reaching humane endpoint. FIG. 4A shows quantified results of the tumor burden assessment (bioluminescence assessment via IVIS) in xenograft mice (Ramos xenograft) post treatment with AAVhu68.Rituximab.coGW in comparison with isotype control. FIG. 4B shows Kaplan-Meier survival analysis of probability of survival in tumor bearing mice (Ramos xenograft) treated with AAVhu68.Rituximab.coGW. These result show that overall survival for AAVhu68.Rituximab.coGW-treated mice was 47.5 days (n=18), in comparison with isotype control-treated mice of 24.5 days (n=12).

[0258] FIG. 5A shows quantified results of the tumor burden assessment (bioluminescence assessment via IVIS, plotted as Total Flux (p / s)) in xenograft mice (OciLy3 xenograft (DLBCL type NHL, with origin from Bone marrow of a 52 year old male patient at relapse)) post treatment with AAVhu68. Rituximab. coGW in comparison with isotype control. FIG. 5B shows Kaplan- Meier survival analysis of probability of survival in tumor bearing mice (OciLy3 xenograft) treated with AAVhu68.Rituximab.coGW. These result show that overall survival for AAVhu68.Rituximab.coGW-trcatcd mice was 48 days (n=15), in comparison with isotypc control-treated mice of 32.5 days (n=14).

[0259] FIG. 6A shows quantified results of the tumor burden assessment (bioluminescence assessment via IVIS) in xenograft mice (Daudi xenograft (Burkitt type NHL, with origin from peripheral blood of a 16 year old male patient)) post treatment with AAVhu68. Rituximab. coGW in comparison with isotype control. FIG. 6B shows Kaplan-Meier surv ival analysis of probability of survival in tumor bearing mice (Daudi xenograft) treated with AAVhu68.Rituximab.coGW. These result show that overall survival for AAVhu68. Rituximab. coGW-treated mice was 55.5 days (n=20), in comparison with isotype control-treated mice of 45 days (n=14).

[0260] Patient-Derived Xenograft Mouse Model of CNSL Remained in Complete Remission (> 21 Weeks) Following ICV Delivery of AAV. Rituximab

[0261] In this study, a patient-derived xenograft (PDX) tissue was purchased from the Dana Farber Cancer Institute, and the patient’s PDX characteristics were provided by the vendor (see Table B below).

[0262] Table B.

[0263] In this study, PDX (PDX Pl) was passaged in-house in Ragl KO mice, with observed tumor growth in 3 out of 5 mice. Then PDX (PDX_P2) from each mouse was dissociated and stored in liquid nitrogen. Following which, 1 vial / mouse were thawed and pooled (n=3), wherein 5 pL of the pooled tumor slurry was implanted in the brain parenchyma in mouse. More specifically, 6-8-week old Ragl KO mice (males and females) were implanted (day 0) with dissociated PDX tissue into the brain parenchyma through the guide-screw' and received IxlO11GC of AAV.Rituximab (AAVhu68. Rituximab. coGW) or AAV.isotype control via ICV injection (day +3 / +4). FIG. 7 shows Kaplan-Meier survival analysis of probability of survival in tumor bearing mice (patient-derived xenograft) treated with AAVhu68.Rituximab.coGW. These result show that overall survival for AAVhu68.Rituximab.coGW-treated mice was not reached (n=10), in comparison with isotype control-treated mice of 12.3 weeks days (n=9). All surviving animals showed no signs of tumor progression. In conclusion, the ICV treatment with AAV. Rituximab led to sustained expression of rituximab at levels sufficient to delay / inhibit xenograft mouse models of CD20+ CNSL. ICV delivery of AAV vectors expressing targeted therapeutics represents a promising treatment strategy to target CNS malignancies.

[0264] All documents cited in this specification are incorporated herein by reference, as is tire Sequence Listing filed herewith, labelled “UPN-23-10397PCT.xmf‘, and the sequences and text therein are incorporated by reference. US Provisional Patent Application No. 63 / 589,612, filed October 11. 2023, and US Provisional Patent Application No. 63 / 643,732, filed May 7, 2024 are incorporated herein by reference. While the invention has been described with reference to particular embodiments, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

CLAIMS:

1. A recombinant adeno-associated virus (rAAV) comprising an adeno- associated virus (AAV) capsid and a nucleic acid molecule comprising an expression cassette in the capsid, wherein the expression cassette comprises:(a) a nucleic acid coding sequence for an anti-human CD20 (hCD20) antibody having a heavy chain and a light chain, the coding sequence comprising:(i) a nucleic acid sequence encoding an IL2 leader peptide operably linked to an anti-hCD20 heavy chain,(ii) the nucleic acid sequence encoding the anti-hCD20 heavy chain comprising SEQ ID NOs:

5.

1. 3 or 7.(iii) a furin cleavage site,(iv) a T2A element linker,(v) a nucleic acid sequence encoding an IL2 leader peptide operably linked to an anti-hCD20 light chain.(vi) the nucleic acid sequence encoding the anti-hCD20 light chain comprising SEQ ID NOs: 6, 2, 4 or 8; and(b) regulatory control sequences operably linked to the sequences of (a), wherein the regulatory sequences comprise a ubiquitin C (UbC) promoter, an optional enhancer, an intron, and a polyadenylation (poly A) sequence.

2. The rAAV according to claim 1, wherein the rAAV comprises nucleic acid molecule comprises a vector genome comprising an AAV 5' inverted terminal repeat (ITR), the expression cassette, and an AAV 3' ITR.

3. The rAAV according to claim 1 or claim 2, wherein the nucleic acid sequence encoding a leader peptide of (i) comprises SEQ ID NO: 20.

4. The rAAV according to any one of claims 1 to 3, wherein the nucleic acid sequence encoding a leader peptide of (v) comprises SEQ ID NO: 21.

5. The rAAV according to any one of claims 1 to 4, wherein the anti-hCD20 heavy chain coding sequence comprises the nucleic acid sequence of SEQ ID NO: 5, or the sequence at least 99% identical to SEQ ID NO: 5 encoding an amino acid sequence of SEQ ID NO: 9, and wherein the anti-hCD20 light chain coding sequence comprises the nucleic acid sequence of SEQ ID NO: 6 or the sequence at least 99% identical to SEQ ID NO: 6 encoding an amino acid sequence of SEQ ID NO: 10.

6. The rAAV according to any one of claims 1 to 5, wherein the anti-hCD20 heavy chain coding sequence comprises the nucleic acid sequence of SEQ ID NO:

5. and wherein the anti-hCD20 light chain coding sequence comprises the nucleic acid sequence of SEQ ID NO: 6.

7. The rAAV according to any one of claims 1 to 6, wherein the anti-hCD20 antibody coding sequence comprises the nucleic acid sequence of SEQ ID NO:

19. or the sequence at least 95% identical to SEQ ID NO: 19.

8. The rAAV according to any one of claims 1 to 4, wherein the anti-hCD20 antibody coding sequence comprises the nucleic acid sequence of SEQ ID NO:

52. the sequence at least 95% identical to SEQ ID NO: 52, SEQ ID NO: 53, the sequence at least 95% identical to SEQ ID NO: 53, SEQ ID NO: 54, or the sequence at least 95% identical to SEQ ID NO: 54.

9. The rAAV according to any one of claims 1 to 8, wherein the UbC promoter has a nucleic acid sequence of SEQ ID NO: 32.

10. The rAAV according to any one of claims 1 to 9, wherein the poly A sequence is SV40 polyA sequence having a nucleic acid sequence of SEQ ID NO: 34.

11. The rAAV according to any one of claims 1 to 10, wherein the intron is a chimeric intron having a nucleic acid sequence of SEQ ID NO: 33.

12. The rAAV according to any one of claims 2 to 11. wherein the AAV ITRs are from AAV2.

13. The rAAV according to any one of claims 1 to 7 and 9-12, wherein the expression cassette comprises the nucleic acid sequence of SEQ ID NO: 28, the nucleic acid sequence at least 99% identical to SEQ ID NO: 28, the nucleic acid sequence of SEQ ID NO: 65, or the nucleic acid sequence at least 99% identical to SEQ ID NO: 65.

14. The rAAV according to any one of claims 2 to 7 and 9-13, wherein the vector genome comprises the nucleic acid sequence of SEQ ID NO: 29 or the sequence at least 99% identical to SEQ ID NO: 29, the nucleic acid sequence of SEQ ID NO: 66, or the nucleic acid sequence at least 99% identical to SEQ ID NO: 66.

15. The rAAV according to any one of claims 1 to 4, 8, and 9-12 wherein the expression cassette comprises the nucleic acid sequence of SEQ ID NOs: 24, 26 or 30.

16. The rAAV according to any one of claims 2 to 4, 8, and 9-12 wherein the vector genome comprises the nucleic acid sequence of SEQ ID NOs: 25, 27 or 31.

17. The rAAV according to any one of claims 1 to 16, wherein the AAV capsid is a clade F AAV capsid.

18. The rAAV according to any one of claims 1 to 16. wherein the AAV capsid is an AAVhu68 capsid.

19. The rAAV according to any one of claims 1 to 16, wherein the AAV capsid is a mutant AAV9 capsid.

20. The rAAV according to any one of claims 1 to 16, wherein the AAV capsid is an AAVrh91 capsid, an AAV9 capsid, a AAVhu95 capsid, an AAVhu96 capsid, or an AAVrh32 / 33 capsid.21 . The rAAV according to any one of claims 1 to 20, which is for use in treating one or more of a CD20-positive B-cell lymphoma, CD20-positive B-cell Non-Hodgkins Lymphoma, a CD20-positive central nervous system (CNS) diffuse large B cell lymphoma (CNS-DLBCL), or CD20-positive B-cell secondary CNS lymphoma (SCNSL).

22. The rAAV according to any one of claims 1 to 20, wherein the rAAV is formulated for systemic and / or central nervous system administration.

23. A composition comprising a stock of rAAV according to any one of claims 1 to 22 and an aqueous suspension media.

24. The composition according to claim 23 wherein the suspension is formulated for intravenous or intrathecal administration, optionally wherein the intrathecal administration is via an intracerebroventricular (ICV) injection or an intracistemal magna (ICM) injection.

25. A pharmaceutical composition comprising a rAAV according to any one of claims 1 to 23 and an aqueous formulation buffer.

26. The pharmaceutical composition according to claim 25, which is formulated for intrathecal administration.

27. The pharmaceutical composition according to claim 25, which is formulated for intracerebroventricular (ICV) injection or intracistemal magna (ICM) injection.

28. A recombinant nucleic acid molecule comprising:(a) an AAV 5' inverted terminal repeat (ITR),(b) an expression cassette comprising a coding sequence for an anti-human CD20 (hCD20) antibody comprising an anti-hCD20 heavy chain and an anti-hCD20 light chain, and regulatory control sequences which are nucleic acid sequences operably linked tothe coding sequence which regulate expression of the anti-hCD20 heavy chain and anti- hCD20 light chain, and(c) an AAV 3' ITR, wherein the expression cassette comprises:(i) an optional enhancer,(ii) a Ubiquitin C (UbC) promoter,(iii) an intron,(iv) a nucleic acid sequence encoding an IL2 leader peptide operably linked to an anti-hCD20 heavy chain,(v) the nucleic acid sequence encoding the anti-hCD20 heavy chain comprising SEQ ID NO: 5, 1, 3 or 7,(vi) a furin cleavage site,(vii) a T2A linker,(viii) a nucleic acid sequence encoding an IL2 leader peptide operably linked to an anti-hCD20 light chain,(ix) the nucleic acid sequence encoding the anti-hCD20 light chain comprising SEQ ID NO: 6, 2, 4 or 8,(x) a polyadenylation (poly A) signal sequence, and wherein the expression cassette further comprises spacer sequences.

29. The recombinant nucleic acid molecule according to claim 28, wherein the expression cassette comprises a nucleic acid sequence of SEQ ID NO: 28.

30. The recombinant nucleic acid molecule according to claim 28, which comprises a nucleic acid sequence of SEQ ID NO: 29.

31. The recombinant nucleic acid molecule according to claim 28, wherein the expression cassette comprises a nucleic acid sequence of SEQ ID NOs: 24, 26 or 30.

32. The recombinant nucleic acid molecule according to claim 28, which comprises a nucleic acid sequence of SEQ ID NOs: 25, 27 or 31.

33. A packaging host cell in culture comprising a recombinant nucleic acid molecule according to any one of claims 28 to 32.

34. The packaging host cell according to claim 33, which further comprises AAV rep coding sequences operably linked to sequences which express rep protein in the packaging host cell, an AAV capsid coding sequences operably linked to sequences which express AAV capsid proteins in the packaging host cell, and helper virus functions necessary to permit packaging of the expression cassette and AAV ITRs into the AAV capsid.

35. The packaging host cell according to claim 33 or 34, wherein the AAV capsid is an AAVhu68 capsid.

36. The packaging host cell according to claim 33 or 34, wherein the AAV capsid is an AAV9 capsid, a mutant AAV9 capsid, an AAVrh91 capsid, an AAVhu95 capsid, an AAVrh32 / 33 capsid, or an AAVhu96 capsid.

37. An rAAV production system useful for producing the rAAV according to any of claims 1 to 20, wherein the production system comprises a cell culture comprising the packaging host cell of any of claims 33 to 36.

38. A method for treating one or more of a CD20-positive B-cell lymphoma, CD20-positive B-cell Non-Hodgkins Lymphoma, a CD20-positive central nervous system (CNS) diffuse large B cell lymphoma (CNS-DLBCL), or CD20-positive B-cell secondary CNS lymphoma (SCNSL), said method comprising administrating to the subject a suspension comprising the rAAV according to any of claims 1 to 20 in a formulation buffer.

39. The method according to claim 38, wherein the suspension is administered intrathecally.

40. The method according to claim 38 or claim 39, wherein said suspension is administered in the absence of chemical or physical disruptors of the blood brain barrier.

41. The method according to claim 38, wherein the suspension is administered via direct injection into tumor bed or via an Ommaya device.

42. A recombinant AAV (rAAV) according to claim 1 to 22 or a composition according to claim 23 or claim 24 for use in preparing a medicament for treatment of one or more of a CD20-positive B-cell lymphoma, CD20-positive B-cell Non-Hodgkins Lymphoma, a CD20-positive central nervous system (CNS) diffuse large B cell lymphoma (CNS- DLBCL), or CD20-positive B-cell secondary CNS lymphoma (SCNSL).

43. An anti -neo plastic regimen comprising administering rAAV according to any one of claims 1 to 22, a composition according to claim 23 or 24. or a pharmaceutical composition according to any one of claims 25 to 27 in combination with a biologic drug, a small molecule, anti-neoplastic agent, radiation, and / or chemotherapeutic agent.

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