Insect cells and methods for modifying same

Abstract

The present disclosure provides insect cell lines for the expression of viral vectors, as well as methods for making and using the same. The provided methods employ the use of identifiers that can be packaged into viral vectors to select and / or identify insect cell lines that have modified sequences associated with beneficial properties for viral vector production. Exemplary viral vectors include AAV vectors.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 315,510, filed March 1, 2022, the disclosure of which is incorporated herein by reference. [Background technology]

[0002] For example, the market for biologics, such as gene therapy and cell therapy, is expanding but is currently burdened by high manufacturing costs and difficulties associated with scaling up production. There is a continuing need for improved cell lines and methods for their isolation for applications including, but not limited to, biologics production and viral vector production. Summary of the Invention

[0003] The present disclosure provides, among other things, methods, systems, and compositions for the production and / or expression of viral vectors in insect cells. The present disclosure recognizes that current technologies for expressing viral vectors in insect cells are burdened by inefficient virus production and screening to isolate optimized insect cell lines. The present disclosure provides platform technologies for modifying insect cells and / or viral vectors for modified characteristics related to viral vector production and other characteristics. In some embodiments, the provided methods enable the production and / or selection of insect cell lines with improved properties for viral vector expression and / or production (e.g., increased viral vector expression, increased expression duration, increased stability, etc.). In some embodiments, the methods include using an identifier to screen viral vectors produced by a library of insect cells. The provided technology includes the surprising insight that by incorporating insect cell identifiers (e.g., including barcode sequences and / or library variants) into and expressing viral vectors, the viral vector production capacity of cells in the library can be efficiently evaluated, characterized, and / or identified. For example, in some embodiments, populations of insect cells are each transformed with a library construct comprising an identifier having an architecture suitable for packaging the identifier into a viral vector (e.g., in the case of an AAV vector, the identifier may be located between the AAV ITRs). In some embodiments, the pool of viral vectors produced by such populations of insect cells can be screened, selected, and / or characterized by the abundance of the unique identifier.

[0004] In some embodiments, an insect cell(s) is provided that comprises one or more modified sequences that together comprise (i) at least one identifier disposed between two viral repeat sequences that can be packaged into a viral vector, and (ii) at least one polynucleotide that comprises one or more nucleic acid sequences essential for the production of a viral vector, wherein the insect cell produces a viral vector that comprises the at least one identifier.

[0005] In some embodiments, the viral vector produced by the insect cell(s) comprises an identifier that is the same as the identifier of the insect cell that produced it. In some embodiments, the identifier of the viral vector is derived from the identifier of the insect cell that produced it. In some embodiments, the identifier of the viral vector corresponds to the identifier of the insect cell that produced it. In some embodiments, the identifier of the viral vector is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the identifier of the insect cell that produced it.

[0006] In some embodiments, an insect cell(s) is provided that comprises one or more modified sequences that together comprise (i) at least one identifier disposed between two viral repeat sequences that can be packaged into a viral vector, (ii) at least one perturbation, and (iii) at least one polynucleotide comprising one or more nucleic acid sequences essential for the production of a viral vector, wherein the insect cell produces a viral vector comprising the at least one identifier. In some embodiments, the insect cell further comprises a payload and / or at least one library variant. In some embodiments, the insect cell further comprises at least one perturbation accessory sequence.

[0007] In some embodiments, an insect cell(s) is provided that comprises one or more modified sequences that together comprise (i) at least one identifier disposed between two viral repeat sequences that can be packaged into a viral vector, (ii) at least one library variant, and (iii) at least one polynucleotide comprising one or more nucleic acid sequences essential for the production of a viral vector, wherein the insect cell produces a viral vector comprising the at least one identifier. In some embodiments, the insect cell further comprises a payload and / or at least one perturbation. In some embodiments, the insect cell further comprises at least one perturbation accessory sequence.

[0008] In some embodiments, an insect cell(s) is provided that comprises one or more modified sequences that together comprise (i) at least one identifier disposed between two viral repeat sequences that can be packaged into a viral vector, (ii) at least one payload, and (iii) at least one polynucleotide comprising one or more nucleic acid sequences essential for the production of a viral vector, wherein the insect cell produces a viral vector comprising the at least one identifier. In some embodiments, the insect cell further comprises at least one library variant and / or at least one perturbation. In some embodiments, the insect cell further comprises at least one perturbation accessory sequence.

[0009] In some embodiments, the insect cell further comprises at least one trans-acting integration sequence and / or at least one cis-acting integration sequence.

[0010] In some embodiments, an insect cell(s) is provided that comprises one or more modified sequences that together comprise: (i) at least one identifier disposed between two viral repeat sequences that can be packaged into a viral vector; (ii) at least one library variant and / or perturbation; (iii) at least one payload; (iv) at least one perturbation accessory sequence; (v) at least one trans-acting integration sequence and / or at least one cis-acting integration sequence; and (vi) at least one polynucleotide comprising one or more nucleic acid sequences essential for the production of a viral vector, wherein the insect cell produces a viral vector comprising the at least one identifier.

[0011] In some embodiments, an insect cell(s) is provided that comprises one or more modified sequences, together comprising (i) at least one identifier disposed between two viral repeat sequences that can be packaged into a viral vector, (ii) at least one polynucleotide comprising one or more nucleic acid sequences essential for the production of a viral vector, and optionally (iii) one or more modified sequences comprising at least one perturbation, at least one library variant, at least one payload, at least one perturbed accessory sequence, at least one trans-acting integration sequence, and / or at least one cis-acting integration sequence, wherein the insect cell produces a viral vector comprising the at least one identifier.

[0012] In some embodiments, an insect cell(s) is provided that comprises (i) a library construct and (ii) one or more modified sequences that together comprise at least one polynucleotide comprising one or more nucleic acid sequences essential for producing a viral vector, wherein the library construct comprises an identifier, the identifier being located between two viral repeat sequences that can be packaged into a viral vector, and the insect cell produces a viral vector comprising the identifier. In some embodiments, the insect cell(s) optionally further comprise one or more modified sequences comprising at least one perturbation, at least one library variant, at least one payload, at least one perturbed accessory sequence, at least one trans-acting integration sequence, and / or at least one cis-acting integration sequence, and the insect cell produces a viral vector comprising the at least one identifier.

[0013] In some embodiments, the viral vector produced by the insect cell(s) comprises the same identifier as the library construct. In some embodiments, the identifier of the viral vector is derived from the identifier of the associated library construct. In some embodiments, the identifier of the viral vector corresponds to the identifier of the library construct. In some embodiments, the identifier of the viral vector is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the identifier of the library construct.

[0014] In some embodiments, an insect cell(s) is provided that comprises (i) a library construct and (ii) one or more modified sequences that together comprise at least one polynucleotide comprising one or more nucleic acid sequences essential for the production of a viral vector, wherein the library construct comprises an identifier, the identifier being located between two viral repeat sequences that can be packaged into a viral vector, and the insect cell produces a viral vector comprising the identifier. In some embodiments, the library construct optionally further comprises one or more modified sequences comprising at least one library variant and / or at least one payload. In some embodiments, the insect cell(s) optionally further comprise one or more modified sequences comprising at least one perturbation, at least one perturbation accessory sequence, at least one trans-acting integration sequence, and / or at least one cis-acting integration sequence, and the insect cell produces a viral vector comprising the at least one identifier.

[0015] In some embodiments, an insect cell population(s) is provided comprising a plurality of insect cells, each individually comprising one or more modified sequences, wherein the one or more modified sequences together comprise (i) at least one identifier disposed between two viral repeat sequences capable of being packaged into a viral vector, and (ii) at least one polynucleotide comprising one or more nucleic acid sequences essential for the production of the viral vector, and wherein the insect cell population produces viral vectors individually comprising the at least one identifier. In some embodiments, the insect cell population optionally further comprises one or more modified sequences comprising at least one perturbation, at least one library variant, at least one payload, at least one perturbed accessory sequence, at least one trans-acting integration sequence, and / or at least one cis-acting integration sequence.

[0016] In some embodiments, an insect cell population(s) is provided that includes a plurality of insect cells, each individually comprising one or more modified sequences, where the one or more modified sequences together comprise: (i) at least one identifier disposed between two viral repeat sequences capable of being packaged into a viral vector; (ii) at least one library variant and / or perturbation; (iii) at least one payload; and (iv) at least one polynucleotide comprising one or more nucleic acid sequences essential for the production of a viral vector, wherein the insect cell population produces viral vectors that individually comprise the at least one identifier. In some embodiments, the insect cell population optionally further comprises one or more modified sequences comprising at least one perturbation accessory sequence, at least one trans-acting integration sequence, and / or at least one cis-acting integration sequence.

[0017] In some embodiments, an insect cell population(s) is provided that includes a plurality of insect cells, each individually comprising one or more modified sequences, wherein the one or more modified sequences together comprise (i) a library construct and (ii) at least one polynucleotide comprising one or more nucleic acid sequences essential for producing a viral vector, wherein the library construct comprises an identifier disposed between two viral repeat sequences that can be packaged into a viral vector, and wherein the insect cell population produces viral vectors that individually comprise the identifiers. In some embodiments, the insect cell population optionally further comprises one or more modified sequences comprising at least one perturbation, at least one library variant, at least one payload, at least one perturbed accessory sequence, at least one trans-acting integration sequence, and / or at least one cis-acting integration sequence.

[0018] In some embodiments, an insect cell population(s) is provided, comprising a plurality of insect cells, each individually comprising one or more modified sequences, wherein the one or more modified sequences together comprise (i) a library construct and (ii) at least one polynucleotide comprising one or more nucleic acid sequences essential for the production of a viral vector, wherein the library construct comprises an identifier disposed between two viral repeat sequences capable of being packaged into a viral vector, and the insect cell population produces viral vectors individually comprising the identifiers. In some embodiments, the library construct optionally further comprises one or more modified sequences comprising at least one library variant and / or at least one payload. In some embodiments, the insect cell population optionally further comprises one or more modified sequences comprising at least one perturbation, at least one perturbation accessory sequence, at least one trans-acting integration sequence, and / or at least one cis-acting integration sequence.

[0019] In some embodiments, the insect cell(s) provided comprise a perturbation (e.g., one or more perturbations). In some embodiments, the insect cell(s) provided produce a viral vector comprising the perturbation (e.g., one or more perturbations). In some embodiments, the insect cell(s) provided comprise a perturbation (e.g., one or more perturbations) that also produces a viral vector comprising the perturbation (e.g., one or more perturbations). In some embodiments, at least one polynucleotide comprising one or more nucleic acid sequences essential for the production of a viral vector comprises a perturbation (e.g., one or more perturbations). In some embodiments, the insect cell(s) provided comprise a perturbation (e.g., one or more perturbations), and at least one polynucleotide comprising one or more nucleic acid sequences essential for the production of a viral vector comprises a perturbation (e.g., one or more perturbations). In some embodiments, the insect cell(s) provided produce a viral vector comprising a perturbation (e.g., one or more perturbations), and at least one polynucleotide comprising one or more nucleic acid sequences essential for the production of a viral vector comprises a perturbation (e.g., one or more perturbations). In some embodiments, the provided insect cell(s) comprise a perturbation (e.g., one or more perturbations) and also produce a viral vector that comprises the perturbation (e.g., one or more perturbations), and at least one polynucleotide comprising one or more nucleic acid sequences essential for production of the viral vector comprises the perturbation (e.g., one or more perturbations).

[0020] The insect cells and / or insect cell populations can include any insect cells known in the art that are suitable for expressing viral vectors. In some embodiments, the insect cells are cells obtained or derived from an armyworm (e.g., Spodoptera frugiperda), a fruit fly (e.g., Drosophila), or a mosquito (e.g., Aedes albopictus).

[0021] In some embodiments, the insect cells comprise Sf21 cells, Sf9 cells, BTI-TN-5B1-4 (High Five) cells, S2 cells, D.Mel2 cells, or any derivative thereof. In some embodiments, the insect cells comprise Sf9 cells and / or derivatives thereof.

[0022] In some embodiments, the insect cells are suitable for suspension cell culture. In some embodiments, the insect cells are suitable for adherent cell culture. In some embodiments, the insect cell population can include suspension cells and / or adherent cells.

[0023] The insect cells and / or methods of the present disclosure may be used to produce any viral vector. In some embodiments, the viral vector produced by the insect cells and / or methods of the present disclosure comprises a perturbation (e.g., one or more perturbations). In some embodiments, the perturbation alters one or more characteristics associated with the production of the viral vector (e.g., viral vector stability) or other characteristics (e.g., altered therapeutic activity).

[0024] In some embodiments, the insect cell(s) of the insect cell library have been modified to destroy or eliminate the receptor(s) for the viral vector(s) being produced, hi some embodiments, the insect cells have been treated with an agent that blocks infection by the viral vector.

[0025] In some embodiments, the viral vector is an adeno-associated virus (AAV) vector, a baculovirus vector, a lentivirus vector, an adenovirus vector, an alphavirus vector, a Sindbis virus vector, a retrovirus vector (e.g., a gammaretrovirus vector), a polyomavirus vector, (e.g., a simian virus 40 (SV40) vector), a papillomavirus vector (e.g., a bovine papillomavirus (BPV) vector), a vaccinia virus vector, a herpes simplex virus (HSV) vector, a measles virus vector, a rhabdovirus vector, a rabies virus vector, a vesicular Stomatitis virus (VSV) vectors, picornavirus vectors (e.g., poliovirus vectors), reovirus vectors, senecavirus vectors, echovirus vectors (e.g., RIGVIR), Semliki Forest virus (SFV) vectors, flavivirus vectors, anellovirus vectors, Newcastle disease virus (NDV) vectors, paramyxovirus vectors, Sendai virus vectors, orthomyxovirus vectors, influenza virus vectors, coronavirus vectors, and / or hybrid virus vectors, and / or derivatives, hybrids, and / or modified derivatives thereof.

[0026] In some embodiments, the insect cells, insect cell populations, and / or methods provided herein are useful for expressing adeno-associated virus (AAV) vectors. In some embodiments, the insect cell(s) comprise (i) at least one identifier disposed between two viral repeat sequences that can be packaged into an AAV vector, and (ii) at least one polynucleotide comprising one or more nucleic acid sequences essential for the production of an AAV vector. In some embodiments, the two viral repeat sequences are each AAV ITR sequences that can be packaged into an AAV vector. In some embodiments, the two viral repeat sequences are each insect AAV ITR sequences. In some embodiments, the two viral repeat sequences are each human AAV ITR sequences.

[0027] In some embodiments, an insect cell population is provided comprising a plurality of insect cells, each of which comprises (i) a nucleic acid sequence comprising a barcode disposed between two functional AAV ITR sequences (the nucleic acid sequence is integrated into the insect genome and disposed between a cis-acting integration sequence pair), (ii) one or more library variants that provide one or more perturbations, and (iii) one or more nucleic acid sequences essential for the production of an AAV vector, wherein the insect cell population produces a plurality of AAV vectors, each AAV vector comprising a barcode corresponding to the barcode of the insect cell that produced it. In some embodiments, at least one library variant comprises a gRNA, and the insect cell(s) further comprise an RNA-guided nuclease. In some embodiments, the AAV vectors produced by the insect cells further comprise a payload.

[0028] In some embodiments, the cis-acting integration sequence of the insect cell(s) is a viral repeat sequence derived from an invertebrate virus (e.g., Junonia coenia densovirus (JcDNV)). In some embodiments, the cis-acting integration sequence of the insect cell(s) is a recombinase recognition site.

[0029] In some embodiments, the one or more perturbations in the insect cell(s) are associated with increased AAV production and / or AAV secretion relative to a reference insect cell population lacking the one or more perturbations, hi some embodiments, the insect cell(s) containing the one or more perturbations exhibit at least a 10% increase in AAV production and / or AAV secretion relative to the reference insect cell lacking the one or more perturbations.

[0030] In some embodiments, the two viral repeat sequences of the insect cells and / or library constructs described herein comprise a pair of inverted terminal repeats (ITRs) that are or include human AAV1 ITR(s), human AAV2 ITR(s), human AAV3b ITR(s), human AAV4 ITR(s), human AAV5 ITR(s), human AAV6 ITR(s), human AAV7 ITR(s), human AAV8 ITR(s), human AAV9 ITR(s), human AAV10 ITR(s), human AAV11 ITR(s), human AAV12 ITR(s), human AAV13 ITR(s), or any combination thereof.

[0031] In some embodiments, the two viral repeat sequences of the insect cells and / or library constructs described herein comprise a pair of inverted terminal repeats (ITRs) that are or include bovine AAV (b-AAV) ITR(s), canine AAV (CAAV) ITR(s), mouse AAV1 ITR(s), caprine AAV ITR(s), rat AAV ITR(s), or avian AAV (AAAV) ITR(s).

[0032] In some embodiments, the insect cell comprises one or more polynucleotides encoding one or more proteins essential for the production of AAV vectors, such as AAV capsid proteins. In some embodiments, the AAV vector comprises human AAV1 capsid protein, human AAV2 capsid protein, human AAV3b capsid protein, human AAV4 capsid protein, human AAV5 capsid protein, human AAV6 capsid protein, human AAV7 capsid protein, human AAV8 capsid protein, human AAV9 capsid protein, human AAV10 capsid protein, human AAV11 capsid protein, human AAV12 capsid protein, or human AAV13 capsid protein. In some embodiments, the AAV vector comprises human ancestral AAV capsid protein. In some embodiments, the AAV vector comprises a bovine AAV (b-AAV) capsid protein, a canine AAV (CAAV) capsid protein, a mouse AAV1 capsid protein, a goat AAV capsid protein, a rat AAV capsid protein, or an avian AAV (AAAV) capsid protein.

[0033] In some embodiments, the insect cells, insect cell populations, and / or methods provided herein are useful for expressing AAV vectors, wherein the insect cell(s) comprise (i) a library construct comprising at least one identifier located between two AAV ITR sequences that can be packaged into an AAV vector, and (ii) at least one polynucleotide comprising one or more nucleic acid sequences essential for AAV vector production, selected from a Rep gene, a Cap gene, a helper gene, or a combination thereof. In some embodiments, the at least one polynucleotide comprising one or more nucleic acid sequences essential for AAV vector formation comprises an AAV Rep gene, an AAV Cap gene, one or more AAV helper genes, or a combination thereof. In some embodiments, the AAV vector is replication-competent. In some embodiments, the AAV vector is replication-conditional, replication-deficient, replication-incompetent, and / or replication-defective.

[0034] In some embodiments, the insect cells, insect cell populations, and / or methods provided herein are useful for expressing baculovirus vectors (e.g., Autographa californica nucleopolyhedrovirus (AcMNPV) vectors).

[0035] In some embodiments, the insect cell, insect cell population, and / or method provided herein are useful for expressing a lentiviral vector. In some embodiments, the insect cell(s) contain (i) at least one identifier located between two viral repeat sequences that can be packaged into a lentiviral vector, and (ii) at least one polynucleotide containing one or more nucleic acid sequences essential for the production of a lentiviral vector. In some embodiments, the two viral repeat sequences are each lentiviral LTR sequences that can be packaged into a lentiviral vector.

[0036] In some embodiments, the two viral repeat sequences of the insect cells and / or library constructs described herein comprise a pair of LTRs, including an HIV LTR, an SIV LTR, an equine infectious anemia virus LTR, an FIV LTR, a Visna virus LTR, or a derivative or combination thereof. In some specific embodiments, the lentiviral vector is an HIV vector and the LTR is an HIV LTR or a derivative thereof. In some embodiments, the lentiviral vector comprises a lentiviral Psi sequence.

[0037] In some embodiments, an insect cell population is provided comprising a plurality of insect cells, each of which comprises (i) a nucleic acid sequence comprising a barcode disposed between two functional lentiviral LTR sequences (the nucleic acid sequence is integrated into the insect genome and disposed between a cis-acting integration sequence pair), (ii) one or more library variants that provide one or more perturbations, and (iii) one or more nucleic acid sequences essential for the production of a lentiviral vector, wherein the insect cell population produces a plurality of lentiviral vectors, each lentiviral vector comprising a barcode corresponding to the barcode of the insect cell that produced it. In some embodiments, at least one library variant comprises a gRNA, and the insect cell(s) further comprises an RNA-guided nuclease. In some embodiments, the lentiviral vectors produced by the insect cells further comprise a payload.

[0038] In some embodiments, the cis-acting integration sequence of the insect cell(s) is a viral repeat sequence derived from a lentivirus. In some embodiments, the cis-acting integration sequence of the insect cell(s) is a recombinase recognition site.

[0039] In some embodiments, the lentiviral LTR comprises an HIV LTR, an SIV LTR, an equine infectious anemia virus LTR, an FIV LTR, a visna virus LTR, or a derivative or combination thereof. In some specific embodiments, the lentiviral vector is an HIV vector, and the LTR is an HIV LTR or a derivative thereof. In some embodiments, the lentiviral vector comprises a lentiviral Psi sequence.

[0040] In some embodiments, the one or more perturbations in the insect cell(s) are associated with increased lentiviral vector production and / or lentiviral vector secretion compared to a reference population of insect cells lacking the one or more perturbations, hi some embodiments, the insect cell(s) containing the one or more perturbations exhibit at least a 10% increase in lentiviral vector production and / or lentiviral vector secretion compared to the reference insect cells lacking the one or more perturbations.

[0041] In some embodiments, the lentiviral vector expressed by the insect cells and methods of the present disclosure is a human immunodeficiency virus (HIV) vector, a simian immunodeficiency virus (SIV) vector, an equine infectious anemia virus vector, a feline immunodeficiency virus (FIV) vector, a visna virus vector, or derivatives thereof.

[0042] In some embodiments, the insect cells comprise one or more polynucleotides encoding one or more proteins essential for the production of lentiviral vectors, such as a lentiviral gag protein or fragments thereof. In some embodiments, the gag protein comprises one or more domains selected from the matrix (MA), capsid (CA), and nucleocapsid (NC) domains. In some embodiments, the insect cells comprise one or more polynucleotides encoding one or more proteins essential for the production of lentiviral vectors, such as a lentiviral envelope protein or fragments thereof.

[0043] In some embodiments, the lentiviral vector is a pseudotyped lentiviral vector comprising gag and envelope proteins from different viruses. In some embodiments, the insect cells comprise one or more polynucleotides encoding one or more proteins necessary for the production of a pseudotyped lentiviral vector comprising gag and / or env proteins from a human immunodeficiency virus (HIV) vector, a simian immunodeficiency virus (SIV) vector, an equine infectious anemia virus vector, a feline immunodeficiency virus vector, a visna virus vector, or derivatives thereof.

[0044] In some embodiments, the insect cells, insect cell populations, and / or methods provided herein are useful for expressing lentiviral vectors, wherein the insect cell(s) comprise (i) a library construct comprising at least one identifier disposed between two lentiviral LTR sequences and a Psi sequence, capable of being packaged into a lentiviral vector, and (ii) at least one polynucleotide comprising one or more nucleic acid sequences essential for the production of a lentiviral vector, selected from a gag gene, an env gene, a pol gene, or a combination thereof. In some embodiments, the at least one polynucleotide comprising one or more nucleic acid sequences essential for the formation of a lentiviral vector comprises a lentiviral gag gene, a lentiviral env gene, a lentiviral pol gene, or a combination thereof. In some embodiments, the lentiviral vector is replication-competent. In some embodiments, the lentiviral vector is replication-conditional, replication-deficient, replication-incompetent, and / or replication-defective.

[0045] In some embodiments, the at least one polynucleotide comprising one or more nucleic acid sequences essential for formation of a lentiviral vector comprises an HIV gag gene, an HIV env gene, an HIV pol gene, or a combination thereof. In some embodiments, the lentiviral vector is a replication-competent HIV vector. In some embodiments, the lentiviral vector is a replication-conditional, replication-deficient, replication-incompetent, and / or replication-defective HIV vector.

[0046] In some embodiments, the insect cells are previously or simultaneously genetically modified to disrupt or eliminate lentiviral receptors. In some embodiments, the insect cells are treated with an agent that blocks lentiviral vector infection. In such embodiments, the integrating vector and / or cis-acting integration sequence is not derived from a lentivirus.

[0047] In some embodiments, the insect cells, insect cell populations, and / or methods provided herein are useful for expressing herpes simplex virus (HSV) vectors. In some embodiments, the insect cell(s) provided comprise (i) at least one identifier disposed between two viral repeat sequences capable of being packaged into an HSV vector, and (ii) at least one polynucleotide comprising one or more nucleic acid sequences essential for the production of an HSV vector. In some embodiments, the HSV vector is or is derived from herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), human cytomegalovirus (HCMV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human herpesvirus 6, and / or human herpesvirus 7, and / or derivatives thereof.

[0048] In some embodiments, the two viral repeat sequences of the insect cells and / or library constructs described herein comprise terminal alpha sequences.

[0049] In some embodiments, the insect cell comprises one or more polynucleotides encoding one or more proteins essential for production of an HSV vector, such as an HSV capsid protein or fragment thereof, hi some embodiments, the HSV capsid comprises VP5, VP19C, VP23, preVP22a, and / or mature protease (the UL26 gene product).

[0050] In some embodiments, the insect cells, insect cell populations, and / or methods provided herein are useful for expressing HSV vectors, wherein the insect cell(s) comprise (i) a library construct comprising at least one identifier disposed between HSV terminal alpha sequences (which sequences can be packaged into an HSV vector), and (ii) at least one polynucleotide comprising one or more nucleic acid sequences essential for the production of an HSV vector comprising an HSV capsid protein.

[0051] In some embodiments, the insect cells, insect cell populations, and / or methods provided herein are useful for expressing HSV-AAV hybrid vectors. In some embodiments, the HSV-AAV hybrid vectors are replication-competent. In some embodiments, the HSV-AAV hybrid vectors are replication-conditional, replication-deficient, replication-incompetent, and / or replication-defective.

[0052] In some embodiments, the provided insect cells and / or populations of insect cells comprise one or more polynucleotides comprising one or more nucleic acid sequences essential for viral vector production. In some embodiments, the polynucleotides comprising one or more nucleic acid sequences essential for viral vector production are present in the insect cell as episomes. In some embodiments, the polynucleotides comprising one or more nucleic acid sequences essential for viral vector production are present in the insect cell genome. In some embodiments, the one or more nucleic acid sequences essential for viral vector production comprise heterologous regulatory elements (e.g., a heterologous promoter and / or a heterologous enhancer). In some embodiments, one or more nucleic acid sequences essential for viral vector production comprise a heterologous promoter sequence that is or includes an SV40 promoter, an elongation factor (EF)-1 promoter, a cytomegalovirus (CMV) promoter, a phosphoglycerate kinase (PGK) 1 promoter, a ubiquitin (Ubc) promoter, a human β-actin promoter, a tetracycline response element (TRE) promoter, a spleen focus-forming virus (SFFV) promoter, a murine stem cell virus (MSCV) promoter, a supercore promoter (SCP), a CAG promoter, or a derivative thereof. In some embodiments, one or more nucleic acid sequences essential for viral vector production comprise a heterologous enhancer sequence that is or includes a CMV early enhancer, a cAMP response element (CRE) enhancer, or a derivative thereof. In some embodiments, one or more nucleic acid sequences essential for viral vector production are under the control of an inducible transcriptional control element. In some embodiments, one or more nucleic acid sequences essential for viral vector production can be integrated into an insect cell genome under the control of an inducible transcriptional control element (e.g., an inducible promoter and / or an inducible enhancer). In some embodiments, one or more nucleic acid sequences essential for the production of a viral vector can be present episomally in insect cells under the control of an inducible transcriptional control element (e.g., an inducible promoter and / or an inducible enhancer).

[0053] Any of the modified sequences of the insect cells and / or populations of insect cells of the present disclosure may be present episomally and / or integrated into the insect cell genome. In some embodiments, one or more modified sequences (including but not limited to, identifiers, perturbations, library variants, payloads, perturbation accessory sequences, trans-acting integration sequences, and / or cis-acting integration sequences) may be present episomally in the insect cell. In some embodiments, one or more modified sequences (including but not limited to, identifiers, perturbations, library variants, payloads, perturbation accessory sequences, trans-acting integration sequences, and / or cis-acting integration sequences) may be integrated into the insect cell genome.

[0054] In some embodiments, one or more modified sequences are present in the viral vector (e.g., identifier, perturbation, payload, etc.). In some embodiments, the insect cell and / or insect cell population expresses a viral vector comprising the identifier and perturbation, respectively. In some embodiments, the viral vector comprises a perturbation that alters one or more characteristics associated with viral vector production and / or other characteristics (e.g., stability, etc.). In some embodiments, the viral vector further comprises a payload. In some embodiments, the viral vector further comprises a reporter and / or a selectable marker.

[0055] In some embodiments, the insect cell comprises a library construct, and one or more polynucleotides comprising the library construct (including but not limited to, identifiers, library variants, payloads, perturbation accessory sequences, trans-acting integration sequences, and / or cis-acting integration sequences) may be present in the insect cell as episomes. In some embodiments, the insect cell comprises a library construct, and one or more polynucleotides comprising the library construct (including but not limited to, identifiers, library variants, payloads, perturbation accessory sequences, trans-acting integration sequences, and / or cis-acting integration sequences) may be integrated into the insect cell. In some embodiments, one or more polynucleotides comprising the library construct may be integrated into the insect cell at a low copy number, for example, four copies or less, three copies or less, two copies or less, or one copy.

[0056] In some embodiments, the one or more modified sequences comprise a heterologous coding sequence. In some embodiments, the one or more modified sequences comprise a heterologous gene and / or a heterologous gene segment. In some embodiments, the one or more modified sequences comprise a heterologous regulatory element (e.g., a heterologous promoter and / or a heterologous enhancer). In some embodiments, the one or more modified sequences comprise a heterologous promoter sequence that is or includes an SV40 promoter, an elongation factor (EF)-1 promoter, a cytomegalovirus (CMV) promoter, a phosphoglycerate kinase (PGK) 1 promoter, a ubiquitin (Ubc) promoter, a human β-actin promoter, a tetracycline response element (TRE) promoter, a spleen focus-forming virus (SFFV) promoter, a murine stem cell virus (MSCV) promoter, a supercore promoter (SCP), a CAG promoter, or a derivative thereof. In some embodiments, the one or more modified sequences comprise a heterologous enhancer sequence that is or includes a CMV early enhancer, a cAMP response element (CRE) enhancer, or a derivative thereof. In some embodiments, the one or more modified sequences comprises an inducible transcriptional control element.

[0057] In some embodiments, the provided insect cells and / or insect cell populations comprise multiple modified sequences (e.g., identifiers, perturbations, library variants, payloads, perturbation accessory sequences, trans-acting integration sequences, and / or cis-acting integration sequences). In some embodiments, the provided insect cells and / or insect cell populations comprise two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more modified sequences. In some embodiments, the insect cells comprise up to 100 modified sequences.

[0058] In some embodiments, the provided insect cells and / or insect cell populations comprise a library construct comprising one or more modified sequences, which may include, for example, an identifier, a library variant, a payload, a perturbed accessory sequence, a trans-acting integration sequence, and / or a cis-acting integration sequence. In some embodiments, the insect cells of the present disclosure comprise at least one library construct, wherein the at least one library construct comprises at least one modified sequence. In some embodiments, the library construct comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more modified sequences. In some embodiments, the library construct comprises up to 100 modified sequences.

[0059] In some embodiments, the provided insect cells and / or populations of insect cells produce viral vectors that include one or more modified sequences, which may include, for example, an identifier, a perturbation, a payload, and / or a cis-acting integration sequence.

[0060] In some embodiments, the insect cells of the present disclosure comprise at least one library construct comprising at least one barcode, at least one identifier, at least one library variant, at least one payload, at least one cis-acting integration sequence, or a combination and / or plurality thereof, and at least one modified sequence selected from the foregoing.

[0061] In some embodiments, the insect cells comprise at least one modified sequence comprising a barcode. In some embodiments, the barcode comprises a sequence that is about 5 to about 25 nucleotides. In some embodiments, the insect cells provided comprise a plurality of unique barcodes, wherein the plurality of unique barcodes comprises unique sequences that are about 5 to about 25 nucleotides.

[0062] In some embodiments, the library construct comprises at least one barcode. In some embodiments, the library construct comprises an identifier comprising at least one barcode. In some embodiments, the library construct comprises an identifier comprising at least one barcode, wherein the barcode is positioned between two viral repeat sequences. In some embodiments, the barcode is positioned between two viral repeat sequences and is not an identifier. In some embodiments, the barcode is not positioned between two viral repeat sequences.

[0063] In some embodiments, the insect cell contains at least one modified sequence, including a library variant. The library variant may include, but is not limited to, a modified sequence including a gene, an ORF, a gRNA sequence, a non-coding nucleic acid, or a combination thereof. In some embodiments, the insect cell contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 library variants. In some embodiments, the insect cell contains up to 100 library variants.

[0064] In some embodiments, the insect cells comprise a plurality of library variants, wherein the plurality of library variants comprises at least one modified sequence comprising at least one unique gene, at least one unique ORF, at least one unique gRNA sequence, and / or at least one unique non-coding nucleic acid, or a combination and / or a plurality thereof. In some embodiments, the insect cells comprise a plurality of library constructs, wherein the plurality of library constructs comprises at least one unique gene, at least one unique ORF, at least one unique gRNA sequence, at least one unique non-coding nucleic acid sequence, or a combination and / or a plurality thereof. In some embodiments, the insect cell population comprises a plurality of library constructs, wherein the plurality of library constructs comprises a plurality of unique genes, a plurality of unique ORFs, a plurality of unique gRNA sequences, a plurality of unique non-coding nucleic acid sequences, or a combination thereof.

[0065] In some embodiments, the library construct comprises a gRNA sequence. In some embodiments, the insect cell population comprises a plurality of library constructs, wherein the plurality of library constructs comprises at least one unique gRNA sequence. In some embodiments, the insect cell population comprises a plurality of library constructs, wherein the plurality of library constructs comprises at least 100 unique gRNA sequences.

[0066] In some embodiments, the library construct comprises an ORF. In some embodiments, the insect cell population comprises a plurality of library constructs, wherein the plurality of library constructs comprises at least one unique ORF. In some embodiments, the insect cell population comprises a plurality of library constructs, wherein the plurality of library constructs comprises at least 100 unique ORFs.

[0067] In some embodiments, the library construct comprises a gene. In some embodiments, the insect cell population comprises a plurality of library constructs, wherein the plurality of library constructs comprises at least one unique gene. In some embodiments, the insect cell population comprises a plurality of library constructs, wherein the plurality of library constructs comprises at least 100 unique genes.

[0068] In some embodiments, the library construct comprises a non-coding nucleic acid sequence. In some embodiments, the insect cell population comprises a plurality of library constructs, each of the plurality of library constructs comprising at least one unique non-coding nucleic acid sequence. In some embodiments, the insect cell population comprises a plurality of library constructs, each of the plurality of library constructs comprising at least 100 unique non-coding nucleic acid sequences.

[0069] In some embodiments, the library construct comprises at least one reporter and / or selectable marker. In some embodiments, one or more polynucleotides comprising the library construct comprise a reporter and / or selectable marker.

[0070] In some embodiments, the library construct comprises an identifier. In some embodiments, the insect cell population comprises a plurality of library constructs, wherein the plurality of library constructs comprises a plurality of identifiers. In some embodiments, the identifier comprises at least one barcode and / or at least one library variant. In some embodiments, the insect cells comprise a library construct comprising an identifier comprising at least one barcode and / or at least one library variant. In some embodiments, the insect cell population comprises a plurality of library constructs comprising a plurality of identifiers, wherein the identifier comprises a plurality of barcodes and / or a plurality of library variants.

[0071] In some embodiments, the library construct comprises a plurality of modified sequences, wherein a first subset of the plurality of modified sequences is positioned between two viral repeat sequences and a second subset of the plurality of modified sequences is positioned outside the two viral repeat sequences.

[0072] In some embodiments, the provided insect cells and / or insect cell populations comprise a plurality of modified sequences comprising at least one library variant and at least one identifier, wherein both the at least one library variant and the at least one identifier are disposed between two viral repeat sequences. In some embodiments, the provided insect cells and / or insect cell populations comprise a plurality of modified sequences comprising at least one library variant, at least one identifier, and at least one payload, wherein the at least one library variant, at least one identifier, and at least one payload are disposed between two viral repeat sequences.

[0073] In some embodiments, the insect cells and / or insect cell populations provided comprise a plurality of modified sequences comprising at least one library variant and at least one identifier, wherein the at least one identifier is located between two viral repeat sequences and the at least one library variant is located outside the two viral repeat sequences. In some embodiments, the insect cells and / or insect cell populations provided comprise a plurality of modified sequences comprising at least one library variant, at least one identifier, and at least one payload, wherein the at least one identifier and the at least one payload are located between two viral repeat sequences and the at least one library variant is located outside the two viral repeat sequences.

[0074] In some embodiments, the provided insect cells and / or populations of insect cells comprise a plurality of modified sequences comprising at least two library variants and at least one identifier, wherein the at least one identifier and at least one library variant of the at least two library variants are positioned between two viral repeat sequences, and at least one library variant of the at least two library variants is positioned outside the two viral repeat sequences. In some embodiments, the provided insect cells further comprise a payload positioned between the two viral repeat sequences.

[0075] In some embodiments, the provided library constructs comprise at least one library variant and at least one identifier, wherein both the at least one library variant and the at least one identifier are disposed between two viral repeat sequences. In some embodiments, the provided library constructs comprise at least one library variant, at least one identifier, and at least one payload, wherein the at least one library variant, at least one identifier, and at least one payload are disposed between two viral repeat sequences.

[0076] In some embodiments, provided library constructs comprise at least one library variant and at least one identifier, wherein the at least one identifier is located between two viral repeat sequences and the at least one library variant is located outside the two viral repeat sequences. In some embodiments, provided library constructs comprise at least one library variant, at least one identifier, and at least one payload, wherein the at least one identifier and the at least one payload are located between two viral repeat sequences and the at least one library variant is located outside the two viral repeat sequences.

[0077] In some embodiments, the provided library construct comprises at least two library variants and at least one identifier, wherein the at least one identifier and at least one library variant of the at least two library variants are positioned between two viral repeat sequences, and at least one library variant of the at least two library variants is positioned outside the two viral repeat sequences. In some embodiments, the provided library construct further comprises a payload positioned between the two viral repeat sequences.

[0078] In some embodiments, the at least one identifier comprises a barcode. In some embodiments, the insect cells and / or library constructs provided comprise at least one modified sequence comprising at least one barcode, wherein the at least one barcode is located between two viral repeat sequences. In some embodiments, the barcode located between the two viral repeat sequences is an identifier. In some embodiments, the insect cells and / or library constructs provided comprise at least one modified sequence comprising at least one barcode, wherein the at least one barcode is located outside the two viral repeat sequences.

[0079] In some embodiments, the insect cells and / or library constructs provided comprise a plurality of barcodes. In some embodiments, the insect cells and / or library constructs provided comprise a plurality of barcodes, at least one barcode being positioned between two viral repeat sequences. In some embodiments, the insect cells and / or library constructs provided comprise a plurality of barcodes, the plurality of barcodes being positioned between two viral repeat sequences. In some embodiments, at least one barcode positioned between two viral repeat sequences is an identifier. In some embodiments, the insect cells and / or library constructs provided comprise a plurality of barcodes, a first subset of the plurality of barcodes being positioned between two viral repeat sequences and a second subset of the plurality of barcodes being positioned outside the two viral repeat sequences.

[0080] In some embodiments, the provided insect cells comprise multiple copies of a library construct or portion thereof. In some embodiments, the provided insect cells comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a library construct or portion thereof. In some embodiments, the provided insect cells comprise multiple copies of a library construct or portion thereof. In some embodiments, the provided insect cells comprise 1 to 4 copies of a library construct or portion thereof. In some embodiments, the provided insect cells comprise exactly 2 copies of a library construct or portion thereof. In some embodiments, the provided insect cells comprise exactly 1 copy of a library construct or portion thereof.

[0081] In some embodiments, the provided insect cells comprise at least one library construct consisting of a single contiguous nucleic acid sequence. In some embodiments, the provided insect cell population comprises a plurality of library constructs, each consisting of a single contiguous nucleic acid sequence, and the plurality of library constructs comprises a plurality of unique nucleic acid sequences. In some embodiments, the library construct comprises a plurality of non-contiguous nucleic acid sequences. In some embodiments, the library construct comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-contiguous nucleic acid sequences. In some embodiments, the library construct comprises up to 100 non-contiguous nucleic acid sequences.

[0082] In some embodiments, the provided insect cell population comprises a plurality of library constructs, each of which is composed of discontinuous nucleic acid sequences, and each of which comprises a plurality of unique nucleic acid sequences. In some embodiments, the nucleic acid or derivative thereof derived from each of the individual insect cells comprises at least one unique cell identity sequence during single-cell sequencing. In some embodiments, the plurality of nucleic acid sequences or derivatives thereof derived from each of the individual insect cells comprises a cell identity sequence during single-cell sequencing. In some embodiments, the nucleic acid or derivative thereof derived from each of the individual library constructs comprises at least one unique cell identity sequence during single-cell sequencing. In some embodiments, the plurality of nucleic acid sequences or derivatives thereof derived from each of the individual library constructs comprises a cell identity sequence during single-cell sequencing.

[0083] In some embodiments, the provided population of insect cells comprises a plurality of library constructs, each insect cell comprising a single library construct consisting of a plurality of discontinuous nucleic acid sequences, the library construct comprising a plurality of unique nucleic acid sequences, and the plurality of nucleic acid sequences (or derivatives thereof) from the library construct comprise a cell identity sequence during single-cell sequencing. In some embodiments, all of the nucleic acid sequences (or derivatives thereof) from the library construct comprise a cell identity sequence during single-cell sequencing.

[0084] In some embodiments, the provided insect cells and / or viral vectors comprise at least one modified sequence comprising at least one perturbation. In some embodiments, the provided insect cells comprise at least one perturbation that is present as an episome in the insect cell. Some provided insect cells comprise at least one perturbation that is present in the genome of the insect cell. In some embodiments, the provided insect cells comprise at least two perturbations, where at least one perturbation is present as an episome and at least one perturbation is present in the genome of the insect cell.

[0085] In some embodiments, the viral vectors expressed by the insect cells provided herein comprise a perturbation (e.g., one or more perturbations). In some embodiments, the viral vectors comprise a modified sequence comprising a perturbation present in the viral nucleic acid. In some embodiments, the viral vectors comprise a modified sequence comprising a perturbation present in the ITRs. In some embodiments, the viral vectors comprise a modified sequence comprising a perturbation present between the ITRs.

[0086] In some embodiments, one or more polynucleotides essential for formation of the viral vector comprise a modified sequence comprising a perturbation (e.g., one or more perturbations). In some embodiments, one or more polynucleotides essential for formation of the viral vector are present as an episome and comprise a modified sequence comprising a perturbation (e.g., one or more perturbations). In some embodiments, one or more polynucleotides essential for formation of the viral vector are present in the genome of an insect cell and comprise a modified sequence comprising a perturbation (e.g., one or more perturbations).

[0087] In some embodiments, the insect cells and / or viral vectors provided comprise multiple unique perturbations, hi some embodiments, the insect cells and / or library constructs provided comprise at least 2, 3, 4, 5, 6, 7, 8, or 9 unique perturbations.

[0088] In some embodiments, the provided insect cells and / or viral vectors comprise at least one perturbation, including an insertion, deletion, substitution, conversion, epigenetic modification, and / or rearrangement, of an endogenous genomic coding sequence. In some embodiments, the endogenous coding sequence is or comprises an endogenous gene or gene segment.

[0089] In some embodiments, the provided insect cells and / or viral vectors comprise at least one perturbation, including an insertion, deletion, substitution, conversion, epigenetic modification, and / or rearrangement, of an endogenous genomic regulatory element. In some embodiments, the endogenous regulatory element is or comprises an endogenous promoter sequence and / or an endogenous enhancer sequence.

[0090] In some embodiments, the provided insect cells comprise at least one perturbation accessory sequence. In some embodiments, the perturbation accessory sequence comprises an RNA-guided nuclease or a derivative thereof. In some embodiments, the RNA-guided nuclease includes Cas9, Cpf1, and / or CasZ, or derivatives thereof, including fusion proteins comprising transcriptional regulators (e.g., Cas9-VPR or Cas9-KRAB-MeCP2 fusions), CRISPR protein fusions with nuclease domains (e.g., Fok1), enzymatic base editors (e.g., BE and ABE fusion versions), reverse transcriptase fusions (e.g., Prime Editor), CRISPR recombinases (e.g., RecCas9), and CRISPR transposases (e.g., Cascade complexes with the Tn7-like transposase system Cas12k and TniQ). In some embodiments, the RNA-guided nuclease comprises Cas9 or a derivative thereof.

[0091] The provided library constructs can be introduced into insect cells using any suitable method known in the art. In some embodiments, the library constructs are introduced into insect cells by transfection and / or transduction. In some embodiments, the library constructs are introduced into insect cells by baculovirus-mediated infection. In some embodiments, the library constructs are introduced into insect cells by lentivirus-mediated transduction.

[0092] In some embodiments, the insect cells provided comprise a library construct, wherein at least one modified sequence of the library construct is present as an episome. In some embodiments, the insect cells provided comprise a library construct, wherein the library construct comprises at least one modified sequence comprising at least one library variant, wherein each individual cell comprises at least one perturbed accessory sequence, and wherein the at least one modified sequence is present as an episome.

[0093] In some embodiments, the provided insect cells comprise a library construct, wherein at least one modified sequence of the library construct comprises at least one library variant, and the at least one library variant is present as an episome. In some embodiments, the at least one library variant is an effector. In some embodiments, the at least one library variant is at least one perturbation. In some embodiments, the at least one library variant comprises at least one ORF, at least one gene, at least one non-coding nucleic acid sequence, and / or at least one gRNA, or a plurality thereof.

[0094] In some embodiments, the provided insect cells comprise a library construct, the library construct comprising at least one modified sequence comprising at least one library variant comprising at least one gRNA, each of the individual cells comprising at least one perturbed accessory sequence comprising an RNA-guided nuclease or a non-RNA-guided nuclease or derivative thereof, and the at least one modified sequence is present as an episome.

[0095] In some embodiments, the provided insect cells comprise a library construct, the library construct comprising at least one modified sequence comprising at least one library variant comprising at least one gRNA, each of the individual cells comprising at least one perturbed accessory sequence comprising an RNA-guided nuclease or a non-RNA-guided nuclease or derivative thereof, and the at least one modified sequence is present as an episome.

[0096] In some embodiments, the provided insect cells and / or insect cell populations comprise at least one perturbation. In some embodiments, the provided insect cells and / or insect cell populations comprise at least one perturbation comprising a genomic sequence alteration, an episomal sequence alteration, and / or an epigenetic modification. In some embodiments, the provided insect cells and / or insect cell populations comprise at least one perturbation comprising an insertion, deletion, substitution, and / or rearrangement of an endogenous genomic coding sequence.

[0097] In some embodiments, the insect cells and / or populations of insect cells provided produce viral vectors that individually comprise at least one perturbation.

[0098] In some embodiments, provided insect cells comprise a library construct, wherein at least one modified sequence of the library construct is present in the genome of the insect cell. In some embodiments, provided insect cells comprise a library construct, wherein the library construct comprises at least one modified sequence comprising at least one library variant, wherein each individual cell comprises at least one perturbed accessory sequence, and wherein the at least one modified sequence is present in the genome of the insect cell.

[0099] In some embodiments, the library construct, or a portion thereof, is inserted into the genome of the insect cell at a random insertion site. In some embodiments, the random insertion site is random within a predetermined subset of genomic locations. In some embodiments, the library construct, or a portion thereof, is inserted into the genome of the insect cell at a predetermined insertion site.

[0100] In some embodiments, the provided insect animal cells and / or library constructs comprise at least one trans-acting integration sequence, wherein the at least one trans-acting integration sequence comprises (i) at least one integration construct, (ii) at least one recombinase and / or its polypeptide, protein, nucleic acid, or polynucleotide product, (iii) at least one nuclease and / or its polypeptide, protein, nucleic acid, or polynucleotide product, (iv) at least one transposase and / or its polypeptide, protein, nucleic acid, or polynucleotide product, and / or (v) at least one modified sequence.

[0101] In some embodiments, the provided insect cells and / or library constructs comprise at least one trans-acting integration sequence comprising an integrating construct. In some embodiments, the provided insect cells and / or library constructs comprise at least one pair of cis-acting integration sequences flanking a first set of viral repeat sequences, and the cis-acting integration sequence comprises a second set of viral repeat sequences. In some embodiments, the integrating construct is an insect viral vector or a derivative thereof.

[0102] In some embodiments, the insect cells and / or library constructs provided comprise a cis-acting integration sequence comprising a first set of viral repeat sequences (e.g., for packaging sequences into a viral vector target) and a second set of viral repeat sequences.

[0103] In some embodiments, the insect cells and / or library constructs provided comprise at least one trans-acting integration sequence that comprises a nuclease and / or its polypeptide, protein, nucleic acid, or polynucleotide product.

[0104] In some embodiments, at least one trans-acting integration sequence comprises at least one nuclease, wherein the at least one nuclease comprises an RNA-guided nuclease or a fusion or derivative thereof. In some embodiments, at least one trans-acting integration sequence comprises at least one nuclease, wherein the at least one nuclease comprises a non-RNA-guided nuclease or a fusion or derivative thereof. In some embodiments, the at least one nuclease comprises Cas9, CasZ, Cpf1, a modified Fok1 nuclease domain fusion with a programmable DNA-binding domain, such as a TALE protein (TALEN) or zinc finger protein (ZFN), and / or a meganuclease, or a derivative thereof. In some embodiments, the at least one nuclease comprises Cas9.

[0105] In some embodiments, the at least one trans-acting integration sequence further comprises at least one modified sequence. In some embodiments, the at least one trans-acting integration sequence comprises an RNA-guided nuclease or a fusion or derivative thereof, and also comprises at least one gRNA.

[0106] In some embodiments, the provided insect cells and / or library constructs comprise at least one pair of cis-acting integration sequences flanking a first set of viral repeat sequences, wherein the cis-acting integration sequences comprise homology arm sequences.

[0107] In some embodiments, at least one trans-acting integration sequence comprises a recombinase and / or its polypeptide, protein, nucleic acid, or polynucleotide product. In some embodiments, the provided insect cells and / or library constructs comprise at least one pair of cis-acting integration sequences flanking a first set of viral repeat sequences, wherein the cis-acting integration sequences comprise a recombinase recognition site. In some embodiments, the recombinase comprises Cre, Flp, Dre, PhiC31, and / or Bxb1, or derivatives thereof.

[0108] In some embodiments, the recombinase comprises a Cre-containing recombinase. In some embodiments, the recombinase recognition sites comprise LoxP sites.

[0109] In some embodiments, the recombinase comprises a Bxb1-containing recombinase. In some embodiments, the recombinase recognition site comprises an Att site.

[0110] In some embodiments, the recombinase comprises an Flp-containing recombinase. In some embodiments, the recombinase recognition site comprises an Frt site.

[0111] In some embodiments, at least one trans-acting integration sequence comprises a transposase and / or its polypeptide, protein, nucleic acid, or polynucleotide product. In some embodiments, the provided insect cells and / or library constructs comprise at least one pair of cis-acting integration sequences flanking a first set of viral repeat sequences, wherein the cis-acting integration sequences comprise a transposase recognition site. In some embodiments, the transposase comprises Piggybac transposase, Sleepingbeauty transposase, and / or Tn5 transposase, or a derivative thereof. In some specific embodiments, the transposase comprises Piggybac transposase or a derivative thereof. In some specific embodiments, the transposase comprises Sleepingbeauty transposase or a derivative thereof. In some specific embodiments, the transposase comprises Tn5 transposase or a derivative thereof.

[0112] In some embodiments, the insect cell population produces a modified viral vector compared to a reference population. In some embodiments, the provided insect cells and / or insect cell populations produce a viral vector that has an altered method of transferring nucleic acid into cells compared to a reference population. In some embodiments, the provided insect cells and / or insect cell populations produce a therapeutically modified viral vector compared to a reference population. In some embodiments, the provided insect cells and / or insect cell populations produce a viral vector that is modified for its intended use compared to a reference population.

[0113] In some embodiments, the provided insect cells and / or insect cell populations produce viral vectors that are less functional in the intended application compared to a reference population. In some embodiments, the provided insect cells and / or insect cell populations produce viral vectors that are non-functional in the intended application compared to a reference population. In some embodiments, the provided insect cells and / or insect cell populations produce viral vectors that are less functional and / or non-functional in introducing nucleic acid into cells compared to a reference population. In some embodiments, the provided insect cells and / or insect cell populations produce viral vectors that are less therapeutically functional and / or non-functional compared to a reference population. In some embodiments, the provided insect cells and / or insect cell populations produce viral vectors that are less functional and / or non-functional in their intended application compared to a reference population.

[0114] In some embodiments, the provided insect cells and / or insect cell populations produce viral vectors that are more functional and / or enhanced in their intended use compared to a reference population. In some embodiments, the provided insect cells and / or insect cell populations produce viral vectors that are more functional and / or enhanced in introducing nucleic acids into cells compared to a reference population. In some embodiments, the provided insect cells and / or insect cell populations produce viral vectors that are more therapeutically functional and / or enhanced compared to a reference population. In some embodiments, the provided insect cells and / or insect cell populations produce viral vectors that are more functional and / or enhanced in their intended use compared to a reference population.

[0115] In some embodiments, the provided insect cells and / or insect cell populations comprise at least one modified sequence (e.g., perturbation) that alters viral vector production under good manufacturing practice compared to a reference cell population. In some embodiments, the provided insect cells and / or insect cell populations comprise at least one modified sequence (e.g., perturbation) that increases viral vector production under good manufacturing practice compared to a reference cell population. In some embodiments, the insect cells with modified viral vector production comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more perturbations compared to the reference cell.

[0116] In some embodiments, the provided insect cells and / or insect cell populations comprise at least one modified sequence (e.g., perturbation) that increases viral vector production under current Good Manufacturing Practice (cGMP). In some embodiments, the at least one modified sequence (e.g., perturbation) increases viral vector production under Good Manufacturing Practice (GMP). In some embodiments, the provided insect cells and / or insect cell populations comprise at least one modified sequence (e.g., perturbation) that increases viral vector production under non-Good Manufacturing Practice (non-GMP).

[0117] In some embodiments, the provided insect cells and / or insect cell populations comprise at least one modified sequence (e.g., perturbation) that increases the viability of the insect cell population compared to a reference cell population. In some embodiments, the provided insect cells and / or insect cell populations comprise at least one perturbation that increases the duration of viral vector production by the insect cell population compared to a reference cell population. In some embodiments, the provided insect cells and / or insect cell populations comprise at least one modified sequence (e.g., perturbation) that increases the genomic stability of the insect cell population compared to a reference cell population.

[0118] In some embodiments, the provided insect cells and / or insect cell populations are produced under pharmaceutical manufacturing standards and contain at least one modified sequence (e.g., perturbation) that reduces the proportion of viral vectors that are less functional in the intended application compared to a reference cell population. In some embodiments, the provided insect cells and / or insect cell populations are produced under pharmaceutical manufacturing standards and contain at least one perturbation that reduces the proportion of viral vectors that are non-functional in the intended application compared to a reference cell population. In some embodiments, the reduced proportion of viral vectors is less functional and / or non-functional in transferring nucleic acid to cells compared to the reference cell population. In some embodiments, the reduced proportion of viral vectors is less therapeutically functional and / or non-functional compared to the reference cell population. In some embodiments, the reduced proportion of viral vectors is less functional and / or non-functional in their intended application compared to the reference cell population.

[0119] In some embodiments, the provided insect cells and / or insect cell populations are produced under pharmaceutical manufacturing standards and contain at least one modified sequence (e.g., perturbation) that increases the proportion of viral vectors that are more functional and / or enhanced in their intended use compared to a reference cell population. In some embodiments, the increased proportion of viral vectors are more functional and / or enhanced in transferring nucleic acid to cells compared to the reference cell population. In some embodiments, the increased proportion of viral vectors are more therapeutically functional and / or enhanced compared to the reference cell population. In some embodiments, the increased proportion of viral vectors are more functional and / or enhanced in their intended use compared to the reference cell population.

[0120] In some embodiments, the at least one modified sequence (e.g., perturbation) increases the proportion of viral vectors under good manufacturing practice that contain all and / or essential nucleic acid sequences and / or other elements for the intended use, compared to a reference cell population.

[0121] In some embodiments, the at least one modified sequence (e.g., perturbation) reduces the proportion of viral vectors under good manufacturing practice that have deleted and / or mutated all and / or essential nucleic acid sequences and / or other elements for the intended use, compared to a reference cell population.

[0122] In some embodiments, the insect cells and / or insect cell populations and / or viral vectors provided comprise a plurality of modified sequences, and the plurality of modified sequences comprises a plurality of perturbations.

[0123] In some embodiments, the reference cell population is (a) an equivalent insect cell population that does not contain at least one modified sequence, and / or (b) a standard cell population capable of producing a viral vector.

[0124] In some embodiments, the viral vectors produced by the insect cells described herein comprise modified sequences (e.g., perturbations) that alter viral vector production under pharmaceutical manufacturing standards compared to a reference cell population. In some embodiments, the viral vector comprises perturbations that alter functionality (e.g., therapeutic function, ability to transfer nucleic acid to cells), genomic stability, production yield in insect cells, duration of production in insect cells, rate of production of viral vector containing all essential nucleic acid sequences, and / or another characteristic relevant to viral vector production, viral vector activity, and / or viral vector use.

[0125] In some embodiments, the insect cell population is produced by (a) introducing a plurality of modified sequences comprising a plurality of library constructs into a plurality of insect cells, each library construct comprising at least one identifier disposed between two first sets of viral repeat sequences, and (b) introducing at least one polynucleotide comprising one or more nucleic acid sequences essential for the production of a viral vector into a plurality of insect cells.

[0126] In some embodiments, the insect cell population is produced by introducing a plurality of modified sequences comprising a plurality of library constructs into a plurality of insect cells, each library construct comprising at least one identifier disposed between two first sets of viral repeat sequences, and the plurality of insect cells comprising one or more nucleic acid sequences essential for production of the viral vector.

[0127] In some embodiments, a method of producing a viral vector is provided, comprising culturing a population of insect cells of the present disclosure under conditions such that the insect cells produce viral vectors, wherein each viral vector produced comprises at least one identifier derived from at least one identifier of the insect cells that produced the viral vector. In some embodiments, each viral vector produced comprises at least one identifier that is identical to at least one identifier of the insect cells that produced the viral vector.

[0128] In some embodiments, a method is provided that includes: (a) producing viral vectors from a library of insect cells, the library of insect cells comprising a plurality of insect cells, each of the plurality of insect cells individually comprising at least one polynucleotide comprising (i) at least one modified sequence, (ii) an identifier, and (iii) one or more nucleic acid sequences essential for production of the viral vector, wherein each viral vector comprises an identifier derived from the identifier of the insect cell that produced the viral vector; and (b) detecting an identifier in the viral vectors. In some embodiments, each viral vector comprises an identifier that is identical to the identifier of the insect cell that produced the viral vector.

[0129] In some embodiments, a method is provided that includes: (a) producing viral vectors from a library of insect cells, the library of insect cells comprising a plurality of insect cells, each of the plurality of insect cells individually comprising at least one polynucleotide comprising (i) at least one modified sequence, (ii) at least one identifier, and (iii) one or more nucleic acid sequences essential for production of a viral vector, wherein each viral vector comprises at least one identifier derived from at least one identifier of the insect cell that produced the viral vector; and (b) detecting the one or more identifiers in the viral vectors by next-generation sequencing. In some embodiments, each viral vector comprises at least one identifier that is identical to at least one identifier of the insect cell that produced the viral vector.

[0130] In some embodiments, a method is provided that includes: (a) generating an AAV library, the AAV library comprising a plurality of AAV vectors derived from a library of insect cells, the library of insect cells comprising a plurality of insect cells, each of the plurality of insect cells individually comprising at least one polynucleotide comprising (i) at least one modified sequence, (ii) at least one barcode sequence, and (iii) one or more nucleic acid sequences essential for production of a viral vector, each AAV viral vector comprising at least one construct, wherein at least one construct comprises a barcode sequence derived from at least one barcode sequence of the insect cells that produced the AAV viral vector; and (b) detecting one or more barcode sequences in the AAV library. In some embodiments, each AAV viral vector comprises at least one construct, wherein at least one construct comprises a barcode sequence identical to at least one barcode sequence of the insect cells that produced the AAV viral vector.

[0131] In some embodiments, a method is provided that includes: (a) generating an AAV library, the AAV library comprising a plurality of constructs derived from a library of insect cells, the library of insect cells comprising a plurality of insect cells, each of the plurality of insect cells individually comprising at least one polynucleotide comprising (i) at least one modified sequence, (ii) at least one barcode sequence, and (iii) one or more nucleic acid sequences essential for production of a viral vector, each AAV viral vector comprising at least one construct, wherein at least one construct comprises a barcode sequence derived from at least one barcode sequence of the insect cells that produced the AAV viral vector; and (b) detecting one or more barcode sequences in the AAV library by next-generation sequencing. In some embodiments, each AAV viral vector comprises at least one construct, wherein at least one construct comprises a barcode sequence identical to at least one barcode sequence of the insect cells that produced the AAV viral vector. In some embodiments, the provided methods further comprise a step of single-cell sequencing of at least one or all nucleic acid sequences or derivatives thereof in each individual cell of the library of insect cells, wherein at least one or all nucleic acid sequences or derivatives thereof comprise a single-cell identity sequence during the single-cell sequencing method, and at least one or all nucleic acid sequences or derivatives thereof comprise at least one library construct.

[0132] In some embodiments, the methods provided herein further comprise single-cell sequencing of at least one or all nucleic acid sequences or derivatives thereof within each individual cell of the library of insect cells, wherein at least one or all nucleic acid sequences or derivatives thereof comprise a single-cell identity sequence during the single-cell sequencing method, and at least one or all nucleic acid sequences or derivatives thereof comprise at least one library variant comprising at least one identifier.

[0133] In some embodiments, the provided methods of the disclosure further comprise introducing a second library construct into an insect cell, the insect cell comprising at least one perturbation derived from the first library construct, the second library construct comprising at least one modified sequence comprising at least one identifier located between the first set of two viral repeat sequences. In some embodiments, a second plurality of library constructs is introduced into a plurality of insect cells, the plurality of insect cells comprising at least one perturbation derived from the first plurality of library constructs, the second plurality of library constructs comprising a plurality of modified sequences, each individual library construct in the second plurality of library constructs comprising at least one identifier located between the first set of two viral repeat sequences. In some embodiments, the provided methods further comprise detecting one or more identifiers and / or one or more modified sequences by single-cell sequencing and / or next-generation sequencing. In some embodiments, the provided methods comprise more than two rounds of introducing the library constructs and detecting one or more identifiers and / or one or more modified sequences. In some embodiments, the provided methods further comprise developing a machine learning model to identify a desired combination of target perturbations using a machine learning approach.

[0134] In some embodiments, the provided methods of the disclosure further comprise removing at least one identifier of the insect cells. In some embodiments, when the provided insect cells and / or library constructs comprise a reporter and / or a selectable marker, the provided methods can comprise removing the reporter and / or selectable marker.

[0135] Throughout the description, when a system or composition is described as having, containing, or comprising particular components, or when a method is described as having, containing, or comprising particular steps, it is contemplated that there are systems or compositions of the invention that consist essentially of or consist of the recited components, and that there are methods of the invention that consist essentially of or consist of the recited steps.

[0136] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0137] The following description is intended only to illustrate and exemplify the present disclosure and is not intended to limit the invention to the specific embodiments described.

[0138] The drawings contained herein, consisting of the following figures, are for illustration purposes only and not for limitation: [Brief explanation of the drawings]

[0139] [Figure 1] 1 shows a schematic diagram of a technology platform for identifying or characterizing viral vector production in insect cells in a library, where the viral vectors incorporate identifiers associated with the insect cells that express them.

[0140] [Figure 2] FIG. 1 shows an exemplary schematic for generating an insect cell library expressing an AAV viral vector containing an identifier.

[0141] [Figure 3-1]

[0023] Figure 1 shows an exemplary schematic for identifying and / or characterizing viral vector production in insect cells in a library by determining the relative enrichment of identifiers in a pool of viral vectors and using single-cell sequencing methods to identify modified sequences in insect cells. [Figure 3-2] Same as above.

[0142] [Figure 4]

[0023] Figure 1 shows a schematic diagram of exemplary library constructs for use in the method for generating an insect cell library expressing AAV viral vectors. pSFX-AAV is an exemplary AAV library construct for episomal expression, and pSFX-PB-AAV is an exemplary AAV in-transposase library construct.

[0143] [Figure 5] 1 shows an exemplary schematic diagram of sequencing an identifier (e.g., barcode) sequence from a viral vector nucleic acid, e.g., a viral vector (e.g., an AAV viral vector) or cellular genomic DNA.

[0144] [Figure 6] FIG. 1 shows an exemplary schematic for generating an insect cell library expressing an AAV viral vector using an AAV-in-BAC library of identifier-containing constructs.

[0145] [Figure 7] FIG. 1 shows an exemplary schematic for generating an insect cell library expressing an AAV viral vector using an AAV-in-transposase library of identifier-containing constructs.

[0146] [Figure 8] 1 shows an exemplary schematic for generating an insect cell library expressing an AAV viral vector using an episomal library of identifier-containing constructs.

[0147] Specific Definitions In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions of these terms and other terms are set forth throughout the specification. Publications and other reference materials referred to herein are incorporated by reference to describe the background of the invention and to provide further detail regarding its practice.

[0148] In this application, unless otherwise clear from the context, (i) the terms "a" and "an" are used herein to refer to one or more (i.e., at least one) objects of a publication; (ii) the term "or" may be interpreted to mean "and / or"; (iii) the terms "comprising" and "including" may be interpreted to encompass itemized elements or steps, whether presented by themselves or with one or more additional elements or steps; and (iv) when ranges are specified, both endpoints are included.

[0149] Throughout this specification, whenever a polynucleotide or polypeptide is represented by a series of letters (e.g., A, C, G, and T for adenosine, cytidine, guanosine, and thymidine, respectively, in the case of polynucleotides), such polynucleotide or polypeptide will be presented in left to right, 5' to 3' or N-terminal to C-terminal order.

[0150] About or Approximately: As used herein, the term "about" or "approximately" can be applied to one or more values ​​of interest, including values ​​similar to the reference value mentioned. Generally, a person skilled in the art familiar with the context will understand the reasonable degree of difference encompassed by "about" or "approximately" in that context. In some embodiments, the term "about" or "approximately" refers to a range of values ​​that fall within ±20% (greater or smaller) of the reference value mentioned, unless otherwise stated or clear from the context. For example, in some embodiments, the term "about" or "approximately" can encompass a range of values ​​that are within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value.

[0151] Amino Acid: As used herein, the term "amino acid" refers to any compound and / or substance that can be incorporated into a polypeptide chain, for example, by the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure, e.g., HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is an unnatural amino acid, in some embodiments, an amino acid is a D-amino acid, and in some embodiments, an amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in natural peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether it is synthetically prepared or obtained from a natural source. As will be clear from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid, and in some embodiments, the term may be used to refer to an amino acid residue of a polypeptide.

[0152] Associated: As used herein, the phrase "associated with" refers to two events or entities when the presence, level, and / or morphology of one is related, connected, or correlated with that of the other. For example, a first entity (e.g., an identifier) ​​is considered to be associated with a second entity (e.g., a modified sequence) when the presence and / or level of the first entity is related to or correlated with the presence and / or level of the second entity (e.g., in insect cells, e.g., through cell division and / or genetic manipulation). In some embodiments, two or more entities are physically "associated" with each other if they are present in the same cell, genome, chromosome, or gene region (e.g., so as to be inherited together through multiple generations of insect cell division). In some embodiments, a particular entity (e.g., an identifier and / or modified sequence) is considered to be associated with a particular phenotype (e.g., intracellular production of viral vectors) when its presence, level, and / or morphology correlates with, for example, an altered level or penetrance of that phenotype (e.g., cells in which the entity exhibits increased viral vector production). In some embodiments, two or more entities are physically associated with one another and interact, directly or indirectly, such that they are in close physical proximity and / or remain in close proximity to one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another. In some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another, but are non-covalently associated, for example, by hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0153] Barcode: As used herein, the term "barcode" refers to any molecular feature (e.g., a nucleic acid sequence) that can substantially distinguish an entity (e.g., a molecule, a nucleic acid, a virus, a cell, etc.) or a combination of entities in a large heterogeneous population of entities. In some embodiments, the barcode is a type of modified sequence. In some embodiments, the barcode is a type of modified nucleic acid sequence. In some embodiments, the barcode is part of a library construct. In some embodiments, the library construct may include multiple barcodes. When a library construct includes one or more barcodes, the one or more barcodes may be included (i) within the identifier, (ii) outside the identifier, or (iii) both within and outside the identifier. In some embodiments, upon detection (e.g., by next-generation sequencing), the one or more barcodes indicate the identity of one or more library variants that are not directly detected (e.g., by next-generation sequencing). In some embodiments, the barcode may include a nucleic acid sequence derived from within a pool of known nucleic acid sequences. In some embodiments, the barcode is an exogenous nucleic acid sequence. In some embodiments, the barcode is an endogenous nucleic acid sequence. In some embodiments, a barcode can include both exogenous and endogenous sequences. Generally, a barcode includes a sequence having a length within a range of 3 nucleotides to 50 nucleotides. For example, in some embodiments, a barcode includes a sequence having a length within a range bounded by a lower limit and an upper limit, where the upper limit is greater than the lower limit. In some embodiments, the lower limit may be about 5 nucleotides, about 6 nucleotides, about 7 nucleotides, about 8 nucleotides, about 9 nucleotides, about 10 nucleotides, about 11 nucleotides, about 12 nucleotides, about 13 nucleotides, about 14 nucleotides, or about 15 nucleotides. In some embodiments, the upper limit may be about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 45 nucleotides, or about 50 nucleotides. In some specific embodiments, a barcode includes a sequence having a length within a range of 5 nucleotides to 25 nucleotides.In some particular embodiments, the barcode comprises a sequence having a length of about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, or about 25 nucleotides.

[0154] Biologic: As used herein, the term "biological" in its broadest sense refers to an item made by or derived from a living organism (e.g., manufactured in a biological system). Biologies according to the present disclosure include, but are not limited to, proteins, polypeptides, nucleic acids, polynucleotides, viruses, viral vectors, therapeutic sera, toxins, antitoxins, vaccines, allergy extracts, blood components or derivatives, gene therapy products, and human tissue or cell products. In some embodiments, a biologic is or comprises a polypeptide-based molecule that can be used to treat, cure, ameliorate, prevent, or diagnose a serious or life-threatening disease or condition. In some embodiments, a biologic is a therapeutic biologic. A therapeutic biologic is a therapeutic biologic that can be applied to prevent, treat, or cure a disease or condition (e.g., in a mammal, e.g., a human). In some embodiments, a biologic is a diagnostic biologic. In some embodiments, a biologic is used in manufacturing (e.g., in the manufacture of a gene therapy or cell therapy product). In some specific embodiments, a biologic is a viral vector.

[0155] Cell identity sequence: As used herein, refers to a nucleic acid sequence for labeling all nucleic acids sequenced from a particular insect cell as part of a single-cell sequencing method. The present disclosure recognizes that single-cell sequencing using cell identity sequences is useful for methods in which the library construct contains multiple discontinuous nucleic acid sequences. In such methods, a given cell identity sequence can provide identifiers and / or associations for all library variants present in the same cell. An exemplary schematic diagram of a method using discontinuous library constructs and single-cell sequencing is shown in Figure 3. However, the present disclosure also recognizes that single-cell sequencing using cell identity sequences can, in some embodiments, also be useful for methods in which the library construct contains a single contiguous nucleic acid sequence. The present disclosure also recognizes that the single-cell identity sequence is specifically added during reverse transcription of expressed RNA during the single-cell sequencing method. It is understood that the construct or nucleic acid for single-cell sequencing should be included in the expressed RNA so that, using appropriate primers during the reverse transcription step, all transcripts are single-cell tagged with the cell identity sequence.

[0156] Cis-acting integration sequence: As used herein, the phrase "cis-acting integration sequence" with respect to a library construct refers to a nucleic acid sequence(s) present in the library construct itself that facilitates integration of one or more trans-acting integration sequences and / or their polypeptide, protein, nucleic acid, or polynucleotide products into a cellular genome (e.g., an insect cell). In some embodiments, the cis-acting integration sequence is included in the library construct and flanks the portion of the library construct that is integrated into the cellular genome. In some embodiments, the cis-acting integration sequence comprises homology arms, recognition sites, and / or viral repeat sequences.

[0157] Equivalent: As used herein, the term "equivalent" refers to two or more agents, entities, circumstances, sets of conditions, subjects, populations, etc. that may not be identical to one another, but are sufficiently similar to permit a comparison between them, where one of skill in the art would understand that conclusions can reasonably be drawn based on observed differences or similarities. In some embodiments, equivalent sets of agents, entities, circumstances, sets of conditions, subjects, populations, etc. are characterized by multiple substantially identical characteristics and one or a few different characteristics. One of skill in the art will understand what degree of identity is required for two or more such agents, entities, circumstances, sets of conditions, subjects, populations, etc. to be considered equivalent in any given situation in context. For example, one skilled in the art will understand that sets of agents, entities, circumstances, sets of conditions, subjects, populations, etc. are equivalent to one another if they are characterized by a sufficient number and variety of substantially identical characteristics to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, stimuli, agents, entities, circumstances, sets of conditions, subjects, populations, etc. are caused by or indicate the existence of differences in various of those characteristics.

[0158] Complementary: As used herein, the term "complementary" refers to nucleotides or nucleotide sequences that base-pair according to the standard Watson-Crick complementarity rules (adenine "A" base-pairs with thymine "T" and guanine "G" base-pairs with cytosine "C"). Nucleotide sequences that exhibit "100% complementary" or "100% complementarity" are nucleotide sequences that base-pair with each other throughout at least one of the two nucleotide sequences. An oligonucleotide can be "100% complementary" to a template polynucleotide that is longer than the oligonucleotide (i.e., an oligonucleotide is "100% complementary" to a template polynucleotide if the entire sequence of the oligonucleotide base-pairs with a portion of the template polynucleotide). However, "complementary" nucleic acid sequences need not be 100% complementary. Generally, the term "complementary" in reference to two or more nucleic acid sequences refers to sufficient complementarity across the two nucleic acid sequences so that they hybridize under stringent conditions and / or temperatures used during the annealing step of an amplification method, e.g., PCR or LCR.

[0159] Construct: As used herein, the term "construct" refers to an entity capable of carrying at least one polynucleotide. In some embodiments, the construct can be a plasmid, transposon, cosmid, or artificial chromosome (e.g., a human artificial chromosome (HAC), yeast artificial chromosome (YAC), bacterial artificial chromosome, or P1-derived artificial chromosome (PAC)). In some embodiments, the construct facilitates the transfer of a polynucleotide into a cell. Certain constructs are capable of autonomous replication within the host cell into which they are introduced (e.g., a plasmid with an origin of replication). Other constructs can be introduced into the genome of a host cell, thereby replicating along with the host genome. In some embodiments, a construct is provided that encodes one or more elements of a viral vector. For example, in some embodiments, a plasmid is provided that encodes one or more elements of a viral vector for expression in insect cells. In some embodiments, a construct is provided that encodes one or more elements of a viral vector. In some embodiments, the construct is a viral vector.

[0160] Control: As used herein, the term "control" refers to the art-known meaning of "control" as a standard against which results are compared. Generally, controls are used to enhance the integrity of experiments by isolating a variable in order to draw conclusions about such variable. In some embodiments, a control is a reaction or assay performed simultaneously with a test reaction or assay to provide a comparator. For example, in one experiment, the "test" (i.e., the variable being tested) is applied. In a second experiment, the "control," i.e., the variable being tested, is not applied. In some embodiments, the control is a pre-existing control (e.g., of a test or assay already performed, or of a previously known amount or result). In some embodiments, the control is or includes a printed or otherwise kept record. In some embodiments, the control is a positive control. In some embodiments, the control is a negative control.

[0161] Corresponding: As used herein with respect to polypeptides and nucleic acids, the term "corresponding" designates the position / identity of an amino acid residue or nucleotide residue, respectively, through comparison to an appropriate reference polypeptide or nucleic acid sequence. For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) can be identified as "corresponding" to a residue in an appropriate reference polymer. For example, for simplicity, one of skill in the art will understand that residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, such that an amino acid "corresponds" to the residue at position 190 (e.g., corresponds to the residue found at position 190 in the reference polypeptide, without necessarily being the 190th amino acid in a particular amino acid chain). One of skill in the art will readily understand how to identify a "corresponding" amino acid (see, e.g., Benson et al. Nucl. Acids Res. (1 January 2013) 41(D1):D36-D42; Pearson et al. PNAS Vol. 85, pp. 2444-2448, April 1988). Those skilled in the art, in conjunction with the present disclosure, will recognize various sequence alignment strategies, including, for example, software programs (e.g., BLAST, FASTA, etc.) that can be utilized to identify "corresponding" residues in polypeptides and / or nucleic acids.

[0162] Endogenous: As used herein, the term "endogenous" refers to anything that exists in its natural context. With respect to a nucleic acid, "endogenous" can refer, for example, to a nucleic acid or sequence thereof that originates within a cell. For example, an endogenous nucleic acid sequence is a sequence that would naturally exist in the genome of an insect cell without human manipulation (e.g., in its natural location, under the control of natural expression element(s)).

[0163] Modified: As used herein, the term "modified" refers to aspects that have been manipulated by the hand of man. With respect to nucleic acids, modification can include any type of modification that can be made to a nucleic acid. In some embodiments, a polynucleotide can be considered "modified" if the sequence has been manipulated by the hand of man to produce a sequence not found in its natural environment, including, for example, deletion, insertion, substitution, conversion, rearrangement, and / or fusion to a second sequence. For example, a modified polynucleotide can contain two or more sequences that are not naturally linked together in that order, but have been manipulated by the hand of man so that in the modified polynucleotide they are directly linked to each other. Similarly, a cell or organism is considered "modified" if it has been manipulated to change its genetic information (e.g., new genetic material not previously present has been introduced, e.g., by transformation, mating, somatic hybridization, transfection, transduction, or other mechanisms, or previously present genetic material has been altered or removed, e.g., by substitution or deletion mutations, or mating protocols). In some embodiments, a modified cell (e.g., a modified insect cell) refers to a cell that has been subjected to manipulation such that its genetic, epigenetic, and / or phenotypic identity is altered relative to an appropriate reference cell, e.g., an otherwise identical cell that has not been so manipulated. In some embodiments, the manipulation is or includes genetic manipulation. In some embodiments, a modified cell is a cell that has been engineered to contain and / or express a particular agent of interest (e.g., a protein, nucleic acid, and / or a particular form thereof) in an altered amount and / or with altered timing relative to such appropriate reference cell. As is common practice and will be understood by those of skill in the art, the progeny of a modified polynucleotide or cell are generally still referred to as "modified," even though the actual manipulation was performed on the earlier entity.

[0164] Modified sequence: As used herein, the phrase "modified sequence" refers to a sequence (e.g., of nucleotides) that has been manipulated by the hand of man. In some embodiments, the modified sequence is a modified nucleic acid sequence. In some embodiments, the modified nucleic acid sequence can include a sequence read in the 5' to 3' direction and / or the 3' to 5' direction. The modified nucleic acid sequence can include any type of modification that can be made to a nucleic acid (e.g., introduction, substitution, deletion, conversion, rearrangement, epigenetic modification, etc.). In some embodiments, the modified sequence is or includes a genetic modification of a genomic sequence of an insect cell. In some embodiments, the modified sequence is or includes an epigenetic modification of genomic DNA of an insect cell. In some embodiments, the modified sequence is or includes an episomal sequence (e.g., introduction or modification of a sequence that exists episomally in a cell). In some embodiments, the modified sequence is or includes an episomal sequence with one or more epigenetic features. In some embodiments, the modified sequence is or includes an identifier. In some embodiments, the modified sequence is or comprises a polynucleotide comprising one or more nucleic acid sequences essential for viral vector production. In some embodiments, the modified sequence is or comprises a perturbation. In some embodiments, the modified sequence is or comprises a library variant. In some embodiments, the modified sequence is or comprises a payload. In some embodiments, the modified sequence is or comprises a perturbation accessory sequence. In some embodiments, the modified sequence is or comprises a trans-acting integration sequence. In some embodiments, the modified sequence is or comprises a cis-acting integration sequence. In some embodiments, the modified sequence is or comprises a library construct. In some embodiments, the modified sequence is or comprises a barcode.

[0165] Episome: As used herein, the term "episome" refers to the extrachromosomal genetic material that can replicate autonomously. In some embodiments, the genetic material (e.g., DNA) as episome can be on a plasmid, cosmid, fosmid, artificial chromosome (e.g., human artificial chromosome (HAC), yeast artificial chromosome (YAC), bacterial artificial chromosome, or P1-derived artificial chromosome (PAC)), or viral vector (e.g., baculovirus vector).

[0166] Exogenous: As used herein, the term "exogenous" refers to any entity that is introduced or has been introduced into an organism or cell. For example, an "exogenous nucleic acid" is a nucleic acid that originates outside of the organism or cell. In some embodiments, an exogenous nucleic acid in an insect cell has been introduced (e.g., by the hand of man) through the cell membrane. In some embodiments, an exogenous nucleic acid can be or include a nucleotide sequence that is present in the native genome in a non-native context (e.g., in a different location and / or under the control of a non-native expression element(s)). In some embodiments, an exogenous nucleic acid can be or include a nucleotide sequence that was not previously present in the genome of the organism or cell (e.g., from a different organism). Exogenous nucleic acids include exogenous genes. An "exogenous gene" is a nucleic acid or sequence thereof that encodes the expression of RNA and / or protein and that has been introduced into an organism or cell (e.g., by transformation / transfection), and is also referred to herein as a "transgene."

[0167] Extrachromosomal: As used herein, the term "extrachromosomal" refers to genetic material that is not contained in a chromosome. With respect to insect cells, chromosomes include nuclear chromosomes and mitochondrial chromosomes. Extrachromosomal genetic material includes episomal genetic material.

[0168] Gene: As used herein, the term "gene" refers to a DNA sequence in a chromosome that encodes a gene product (e.g., an RNA product, e.g., a polypeptide product). In some embodiments, a gene comprises coding sequence (i.e., a sequence that encodes a specific product). In some embodiments, a gene comprises non-coding sequence. In some particular embodiments, a gene may include both coding (e.g., exons) and non-coding (e.g., intron) sequences. In some embodiments, a gene may include one or more regulatory sequences (e.g., promoters, enhancers, etc.) and / or intron sequences that, for example, can control or influence one or more aspects of gene expression (e.g., cell-type specific expression, inducible expression, etc.). As used herein, the term "gene" generally refers to a portion of a nucleic acid that encodes a polypeptide or fragment thereof; the term may optionally encompass regulatory sequences, as will be clear to one of skill in the art from the context. This definition is not intended to exclude the application of the term "gene" to non-protein-coding expression units, but it is intended to clarify that in most cases, the term as used herein refers to a nucleic acid that encodes a polypeptide. In some embodiments, a gene may encode a polypeptide, but the polypeptide may not be functional; for example, a gene variant may encode a polypeptide that does not function in the same way, or at all, compared to the wild-type gene.

[0169] Genome: As used herein, the term "genome" refers to the entire genetic information carried by an individual organism or cell, as represented by the complete nucleic acid sequences of its chromosomes.

[0170] Guide sequence: As used herein, the term "guide sequence" refers to a nucleic acid sequence that corresponds to the nucleic acid sequence of a guide RNA for nuclease-mediated editing (e.g., by an RNA-guided nuclease). The terms "guide RNA" and "gRNA" refer to any nucleic acid that facilitates the specific association (i.e., "targeting") of an RNA-guided nuclease, such as Cas9 or Cpf1, with a target sequence, such as a genomic or episomal sequence within a cell. A gRNA can be unimolecular (comprising a single RNA molecule, or called a chimera) or modular (often comprising multiple, usually two separate RNA molecules, e.g., crRNA or tracrRNA, associated with each other, e.g., by duplexing). Whether unimolecular or modular, a guide RNA comprises a "guide sequence" that is fully or partially complementary to a sequence within a target, such as a DNA sequence within the genome of a cell where editing is desired. Guide sequences are referred to by various names in the literature, including, but not limited to, "targeting domain," "complementarity region" (e.g., Cotta-Ramusino et al., WO 2016 / 073990), "spacer" (e.g., Briner et al., Molecular Cell 56(2), 333-339, October 23, 2014), and generally "crRNA" (e.g., Jiang et al. Nat Biotechnol. 2013 Mar;31(3):233-239). Regardless of the name they are given, guide sequences are typically about 10-30 nucleotides in length. In some embodiments, guide sequences are 15-25 nucleotides in length. In particular embodiments, guide sequences are 16-24 nucleotides in length (e.g., 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides in length). In some embodiments, the guide sequence is at or proximal to the 5' end of the gRNA (e.g., with Cas9 or a nuclease derived or derived therefrom), In some embodiments, the guide sequence is at or proximal to the 3' end of the gRNA (e.g., with Cpf1 or a nuclease derived or derived therefrom).

[0171] Heterologous: As used herein, the term "heterologous" refers to any entity that does not naturally occur in the particular context (e.g., cell or organism) in which it is present. With respect to nucleic acids, heterologous refers to sequences that do not naturally occur together (e.g., in the same polynucleotide or the same cell). In some embodiments, a heterologous sequence can be a rearrangement, conversion, insertion, or substitution of a sequence into a non-endogenous context (e.g., from a different genomic location or from a different organism). For example, a heterologous promoter sequence or a heterologous enhancer sequence can be a naturally associated sequence from a different gene or a different organism. In some embodiments, the heterologous sequence is present in the genome of an insect cell. In some embodiments, the heterologous sequence is present as an episome in the insect cell.

[0172] Homology: As used herein, the term "homology" or "homologue" refers to the overall relatedness between oligonucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. In some embodiments, oligonucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be "homologous" to one another if their sequences are at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, oligonucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., contain residues with related chemical properties at corresponding positions). For example, as is well known to those skilled in the art, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids and / or as having "polar" or "nonpolar" side chains, as being similar to one another. Substitution of one amino acid for another amino acid of the same type may often be considered a "homologous" substitution.

[0173] Identifier: As used herein, refers to an element that (i) can be detected (e.g., by next-generation sequencing) and (ii) can identify the insect cell or clonal cell line from which the viral vector was produced and / or derived. The present disclosure provides identifiers that are shared and / or transferred between insect cells and viral vectors. In some embodiments, the identifier comprises a nucleic acid sequence. In some embodiments, the identifier comprises one or more barcodes, one or more library variants, or a combination thereof. In some embodiments, the identifier comprises a sequence that can be detected, for example, by PCR (e.g., quantitative PCR), hybridization (e.g., using a probe), and / or sequencing (e.g., next-generation sequencing and / or Sanger sequencing). In some embodiments, the identifier is or comprises a nucleic acid sequence that is shared and / or transferred between an insect cell and a viral vector. In some embodiments, the identifier is included in a library construct (e.g., for introduction into an insect cell). In some embodiments, the identifier is included in a library construct among genetic architectures suitable for packaging the identifier into a viral vector (e.g., viral repeat sequences, e.g., AAV ITR sequences). In some embodiments, the identifier is present within the genome of the viral vector (e.g., between viral repeat sequences). In some embodiments, detection of the identifier in the genome of the viral vector (e.g., between viral repeat sequences) is associated with the insect cell or clonal cell line in which the viral vector was produced or derived. In some embodiments, detection of the identifier in the genome of the viral vector (e.g., between viral repeat sequences) is associated with the presence of one or more library constructs and / or one or more library variants associated with the insect cell or clonal cell line in which the viral vector was produced or derived. In some embodiments, the provided methods can include sequencing the identifier (e.g., using next-generation sequencing) to determine the relative abundance of a particular viral vector in a pool or sample of viral vectors.

[0174] Identity: As used herein, the term "identity" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison (e.g., for optimal alignment, gaps can be introduced into one or both of the first and second sequences, and non-identical sequences can be ignored for comparison purposes). In some embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence, and nucleotides at corresponding positions are then compared. If a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the two molecules (i.e., the first and second) are identical at that position. The percentage of identity between two sequences is a function of the number of identical positions shared by the two sequences being compared, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Sequence comparison and determination of the percentage of identity between two sequences can be achieved using a mathematical algorithm. For example, the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17, incorporated in its entirety by reference) incorporated into the ALIGN program (version 2.0) can be used to determine the percentage of identity between two nucleotide sequences.

[0175] Integrating construct: As used herein, refers to a construct that facilitates genomic integration of a construct, such as a library construct. In some embodiments, the integrating construct is an insect viral vector (e.g., Junonia coenia densovirus (JcDNV) or a derivative thereof). In some embodiments, the integrating construct comprises a cis-acting integration sequence and / or a trans-acting integration sequence.

[0176] Isolated: As used herein, refers to substances and / or entities that are (1) separated from at least some of the components with which they were associated when originally produced (whether in nature and / or in an experimental setting) and / or (2) designed, produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were originally associated. In some embodiments, an isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may be considered "isolated" or even "pure" even after being combined with certain other components, such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.). In such embodiments, the isolation or purity of a substance is calculated without including such carriers or excipients. By way of example only, in some embodiments, a naturally occurring biological polymer, such as a polypeptide or polynucleotide, is considered "isolated" if: a) by reason of its origin or source, it is not associated with some or all of the components that naturally accompany it in nature; b) it is substantially free from other polypeptides or nucleic acids of the same species that produces it in nature; or c) it is expressed by or is otherwise associated with components of a cell or other expression system other than the species that produces it in nature. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system other than the one that produces it in nature is considered to be an "isolated" polypeptide.Alternatively, or in addition, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered to be an "isolated" polypeptide so long as it has been separated from a) other components with which it is associated in nature, and / or b) other components with which it was associated when originally produced.

[0177] Library construct: As used herein, refers to one or more nucleic acid constructs for generating an insect cell library. As used herein, a library construct includes (i) at least one identifier, (ii) a genetic architecture suitable for packaging the identifier into a viral vector (e.g., viral repeat sequences, e.g., AAV ITR sequences), and (iii) any library variants, if not already included within the identifier. A library construct may also include other modified sequences, such as, but not limited to, any cis-acting integration sequences (if genomic integration of the library construct is relevant) and / or any barcodes. In some embodiments, all elements of a library construct are included in a single (i.e., one) contiguous piece of nucleic acid (e.g., DNA). In some embodiments, a library construct refers to multiple separate, discontinuous pieces of nucleic acid (e.g., DNA). When a library construct includes multiple nucleic acids (i.e., a discontinuous library construct), it includes at least one construct including an identifier (e.g., viral repeat sequences, e.g., AAV ITR sequences) positioned between sequences for packaging the identifier into a viral vector. In some embodiments where the library construct is discontinuous, the library construct further comprises one or more constructs, each of which comprises one or more library variants. The provided methods using discontinuous library constructs also include identifying one or more library variants of the library construct (e.g., single-cell sequencing of insect cells). For example, Figure 3 illustrates a method using a library construct comprising separate pieces of DNA, showing a cell containing a construct comprising an identifier (e.g., a barcode flanked by ITRs) and three additional constructs comprising library variants (e.g., three separate, unique gRNAs), where the identifiers (e.g., barcodes) are packaged into a viral vector and the associations with the library variants are determined by single-cell sequencing. While single-cell sequencing is not necessary in many methods using a single, contiguous library construct, it may still be useful in some circumstances.

[0178] Library variant: As used herein, refers to an element of a library construct that creates a perturbation that varies between cells. For example, in a population of cells, each cell obtains a different library construct containing a unique library variant or combination of library variants. For example, a library variant may include a gene, an ORF, a gRNA sequence, a non-coding nucleic acid, or multiple and / or combinations thereof. For the purposes of the present disclosure, a library variant is separate from a barcode, although one or more library variants can be associated with one or more barcodes. In some embodiments, one or more library variants and / or barcodes can be included within or outside an identifier within a library construct. In some embodiments, one or more barcodes themselves can be directly detected (e.g., in next-generation sequencing methods), rather than the library variant itself. In some embodiments, a library variant may be referred to herein as an effector, whereby the library variant affects or results in a perturbation that varies between cells. In some embodiments, the library variant itself can be a perturbation that varies between cells. For example, in some embodiments, library variants that are gRNAs are effectors that, together with an RNA-guided nuclease (e.g., a perturbed accessory sequence), effect a deletion in the genomic DNA of a cell. In other embodiments, library variants that are ORFs or gene sequences, upon transfection into a cell and, in some cases, integration into genomic DNA (e.g., carried out by trans- and cis-acting integration sequences), themselves result in perturbations or modifications of the genetic material of a cell.

[0179] Nucleic Acid: As used herein, in its broadest sense, refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, nucleic acids are compounds and / or substances that are or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As is clear from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides), and in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" is or comprises RNA, and in some embodiments, "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs.

[0180] Payload: As used herein, the term "payload" refers to any entity of interest for delivery by a viral vector produced by the methods of the present disclosure. For example, it may be desirable to introduce such a payload into a cell, organ, organism, and / or biological system (e.g., containing a cell). In some embodiments, the payload sequence is or includes a heterologous nucleic acid sequence for delivery by a viral vector of the present disclosure. In some embodiments, the payload sequence includes one or more of a coding region, a gene regulatory element, and a transcription terminator. Non-limiting examples of gene regulatory elements include a promoter, a transcription activator, an enhancer, and a polyadenylation signal. In some embodiments, the payload sequence includes a coding region, a gene regulatory element, and a transcription terminator, which are arranged relative to each other such that the coding region is between the gene regulatory element and the transcription terminator. In some embodiments, the coding region encodes a gene product. In some embodiments, the gene product is RNA. In some embodiments, the coding region encodes a polypeptide (e.g., a protein, e.g., a glycoprotein). In some embodiments, the coding region encodes a fusion polypeptide and / or a chimeric polypeptide. In some embodiments, the coding region encodes one gene product. In some embodiments, the coding region encodes multiple gene products (e.g., 2, 3, 4, 5, 6, 7 or more gene products). In some embodiments, the coding region encodes a regulatory RNA (e.g., siRNA, microRNA, etc.). In some specific embodiments, the payload encodes one or more entities for gene editing (e.g., a gRNA-mediated editing system). In some embodiments, the payload encodes a protein product.

[0181] Perturbation: As used herein, refers to a genetic modification in an insect cell, a viral vector produced, a polynucleotide essential for the production of a viral vector, and / or other constructs that results from and / or is identified by the methods described herein. In some embodiments, the insect cell, the viral vector produced, the polynucleotide essential for the production of a viral vector, and / or other constructs contain one or more perturbations that are generated, produced, identified, and / or selected from the insect cell libraries of the present disclosure for the expression and / or production of a viral vector. In some embodiments, the perturbation is the result of one or more library variants. In some embodiments, the perturbation is a genetic modification that results from and / or is identified by the methods described herein, rather than the result of a library variant. In some embodiments, the perturbation comprises a genetic modification in at least one polynucleotide that includes one or more nucleic acid sequences essential for the production of a viral vector. In some embodiments, the perturbation is associated with one or more desired characteristics of the insect cell (e.g., for the expression of a viral vector (e.g., independently and / or synthetically)) or the viral vector. In some embodiments, the perturbation includes a genomic sequence change (e.g., a genomic insertion, deletion, substitution, rearrangement, etc.), an episomal sequence change, and / or an epigenetic modification.

[0182] Perturbation accessory sequence: As used herein, it includes any sequence that helps to create perturbation in combination with a library construct. For example, in some embodiments, the library construct includes a library variant that includes a gRNA, and the perturbation accessory sequence includes a sequence that encodes an RNA-guided nuclease or other element for nuclease-mediated perturbation.

[0183] Polypeptide: As used herein, the term "polypeptide" refers to any polymeric chain of residues (e.g., amino acids) typically linked by peptide bonds. In some embodiments, a polypeptide has a naturally occurring amino acid sequence. In some embodiments, a polypeptide has a non-naturally occurring amino acid sequence. In some embodiments, a polypeptide has an amino acid sequence that has been modified so as to be designed and / or produced by the hand of man. In some embodiments, a polypeptide may comprise or consist of naturally occurring amino acids, non-naturally occurring amino acids, or both. In some embodiments, a polypeptide may comprise one or more pendant groups or other modifications, e.g., modifications of or attachment to one or more amino acid side chains, at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications may be acetylation, amidation, lipidation, methylation, pegylation, etc., or combinations thereof. In some embodiments, a polypeptide may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known to those of skill in the art. In some embodiments, useful modifications may be or include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins can include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and / or characteristic portions thereof.

[0184] Polynucleotide: As used herein, the term "polynucleotide" refers to any polymeric chain of nucleic acid. In some embodiments, a polynucleotide is or comprises RNA, and in some embodiments, a polynucleotide is or comprises DNA. In some embodiments, a polynucleotide is, comprises, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a polynucleotide is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, polynucleotide analogs differ from nucleic acids in that they do not utilize a phosphodiester backbone. Alternatively or additionally, in some embodiments, a polynucleotide has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, a polynucleotide is, comprises, or consists of one or more naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, a polynucleotide is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some embodiments, polynucleotides comprise one or more modified sugars (eg, 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to the sugars of natural nucleic acids.In some embodiments, the polynucleotide has a nucleotide sequence that encodes a functional gene product, such as RNA or a protein. In some embodiments, the polynucleotide comprises one or more introns. In some embodiments, the polynucleotide is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), replication in a recombinant cell or system, and chemical synthesis. In some embodiments, a polynucleotide is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues in length. In some embodiments, the polynucleotide is partially or entirely single-stranded, and in some embodiments, the polynucleotide is partially or entirely double-stranded. In some embodiments, the polynucleotide has a nucleotide sequence that includes at least one element that encodes or is the complement of a sequence that encodes a polypeptide. In some embodiments, the polynucleotide has enzymatic activity.

[0185] Predetermined: As used herein, the term "predetermined" refers to prior to the initiation of an experiment and / or analysis. For example, in some embodiments, the location or characteristics of a modified sequence may be considered predetermined if a set of expected outcomes (e.g., insertion site) is known prior to the physical act of introducing the modified sequence (e.g., the modified sequence may be inserted into one of several different genomic locations).

[0186] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a series of at least two amino acids linked together by peptide bonds). A protein may contain moieties other than amino acids (e.g., may be a glycoprotein, proteoglycan, etc.) and / or may be processed or modified in other ways. Those of skill in the art will understand that a "protein" may be the entire polypeptide chain (with or without a signal sequence) produced by a cell, or a characteristic portion thereof. Those of skill in the art will understand that a protein may in some cases comprise multiple polypeptide chains linked, for example, by one or more disulfide bonds or associated by other means.

[0187] Reference: As used herein, the term "reference" refers to a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value, respectively. In some embodiments, the reference or control is tested and / or determined substantially contemporaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Generally, as will be understood by those of skill in the art, a reference or control is determined or analyzed under conditions or circumstances comparable to those being evaluated. Those of skill in the art will understand when there is sufficient similarity to justify reliance on and / or comparison to a particular reference or control being considered. In some embodiments, the reference is a negative control reference; in some embodiments, the reference is a positive control reference. In some embodiments, the comparison is made to a reference cell or reference cell population with comparable genetic characteristics and cultured under comparable conditions (except with respect to the variable being analyzed). In some particular embodiments, the reference cells differ with respect to the presence of at least one modified sequence and / or at least one barcode sequence, but are otherwise identical.

[0188] RNA-guided nuclease: As used herein, the term "RNA-guided nuclease" refers to a polypeptide that binds to and is directed to a specific target nucleotide sequence in a sequence-specific manner by a guide RNA molecule (complexed with a polypeptide and hybridized to the target sequence). While an RNA-guided nuclease can cleave a target sequence upon binding, the term RNA-guided nuclease also encompasses nuclease-inactive RNA-guided nucleases that can bind to but not cleave a target sequence. Cleavage of a target sequence by an RNA-guided nuclease can result in single-stranded or double-stranded cleavage. RNA-guided nucleases that can cleave only one strand of a double-stranded nucleic acid molecule are referred to herein as nickases. In some embodiments, the RNA-guided nuclease is or is derived from Cas9, CasZ, Cpfl, and / or Fok1.

[0189] Trans-acting integration sequence: As used herein, the phrase "trans-acting integration sequence" with respect to a library construct refers to a nucleic acid sequence, not necessarily included as part of the library construct itself, required for integration of the library construct into an insect cell genome. The trans-acting integration sequence and / or its polypeptide, protein, nucleic acid, or polynucleotide product (e.g., a recombinase) cooperates with a cis-acting integration sequence (e.g., a recombinase site) to effect integration of the library construct, or a portion thereof, into the cellular genome (e.g., an insect cell). In some embodiments, integration of a library construct requires multiple trans-acting integration sequences.

[0190] Transformation or transfection: As used herein, the terms "transformation" or "transfection" refer to any process of introducing foreign DNA into a host cell (e.g., an insect host cell). Transformation can occur under natural or artificial conditions using a variety of methods well known in the art. Transformation can rely on any known method for the insertion of foreign nucleic acid sequences into eukaryotic host cells. In some embodiments, the specific transformation methodology is selected based on the host cell to be transformed and may include, but is not limited to, viral infection or transduction, electroporation, and lipofection. In some embodiments, a "transformed" cell is stably transformed, in that the inserted DNA is capable of replicating as an autonomously replicating plasmid or as part of the host chromosome. In some embodiments, the transformed cell transiently expresses the introduced nucleic acid for a limited period of time.

[0191] Viral Vector: As used herein, the term "viral vector" refers to an entity that (i) can carry at least one polynucleotide and (ii) comprises viral proteins (e.g., capsid proteins, e.g., viral capsid proteins and / or variants or derivatives thereof). In some embodiments, a viral vector comprises one or more nucleic acid molecules. In some embodiments, a viral vector can facilitate transfer of nucleic acid into a cell. In some embodiments, a viral vector comprises one or more nucleic acid sequences and one or more viral capsid proteins. In some embodiments, a viral vector comprises capsid proteins and / or nucleic acid sequences from an adeno-associated virus (AAV) vector, an adenovirus vector, a baculovirus vector, a lentivirus vector, and / or a retrovirus vector. In some embodiments, a viral vector comprises an envelope.

[0192] All literature and similar materials cited in this application, regardless of format, including, but not limited to, patents, patent applications, articles, books, papers, and web pages, are expressly incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differs from or conflicts with this application (including, but not limited to, defined terms, term usage, techniques described, etc.), this application controls. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described in any way. DETAILED DESCRIPTION OF THE INVENTION

[0193] Biologics, including cell and gene therapies, vaccines, and biological reagents, are a rapidly growing market. Insect cells can efficiently express recombinant proteins and can be used to produce several biologic products, including viruses, virus-like particles, and / or vaccines. However, the supply chain for producing biologics, especially those that use viral vectors, is highly inefficient. Even subtle changes in the viral vector or insect host cell can affect manufacturing yield. For example, the payload and serotype of the viral vector can affect insect cell metabolism, viability, viral vector assembly, viral vector production, and / or viral vector expression. Biological production of viral vectors, such as in insect cell culture, is important for reducing costs and complying with good manufacturing practices. Insect cells contain most of the post-translational modification pathways present in mammalian systems, allowing for the production of biologic products that are more similar (e.g., antigenically, immunogenically, and / or functionally) to native mammalian proteins than those expressed in yeast or prokaryotic cells. The present disclosure recognizes the problem with viral vector production in that insect cell lines are generally not optimized and / or screening platforms for isolating optimized cell lines are inefficient. Current manufacturing of viral vectors is inadequate due to both the high cost and long production times of biological production and the increasing market demand for large quantities of viral vectors.

[0194] The present disclosure provides, among other things, platform technologies for expressing viral vectors in insect cells. The disclosure provides methods using a library-based approach to screen, evaluate, and / or characterize insect cells or cell lines based on their ability to express viral vectors. The provided technology allows viral vectors to incorporate identifiers (e.g., including barcode sequences and / or library variants) for the insect cells expressing them, thereby providing an efficient and robust method for sequencing the viral vector genome (e.g., among viral repeat sequences) and identifying insect cells with higher viral vector expression and / or other characteristics of viral vector production. The provided methods, systems, and compositions provide a rapid and robust platform for modifying insect cells with beneficial viral vector properties (e.g., increased viral vector expression, increased expression duration, increased stability, etc.).

[0195] Insect cell platform for viral vector expression The present disclosure provides a platform technology for producing, screening, selecting, modifying, and / or identifying insect cell(s) or cell line(s) for expression of viral vectors. The present disclosure provides methods using a library-based approach with an identifier indicating the insect cell origin within the library. The present disclosure provides a novel platform technology in which viral vectors expressed by insect cells incorporate an identifier (e.g., comprising a barcode and / or library variant). Viral vectors produced from an insect cell library can be pooled and analyzed for desired properties (e.g., by sequencing, e.g., by next-generation sequencing). Incorporating the identifier into the viral vector allows for the rapid identification of insect cells producing viral vectors with desired characteristics, such as, but not limited to, enhanced or improved viral vector production. The present disclosure encompasses the recognition that an insect cell library can be screened for cell lines with desired characteristics for viral vector production (e.g., improved viral vector production) by sequencing the viral vector's identifier (e.g., comprising a barcode and / or library variant). For example, the relative abundance of a particular identifier in a pool of identifiers derived from viral vectors expressed by an insect cell library can correlate with the relative viral vector productivity of a particular insect cell line in the library (e.g., the presence of a particular identifier in high abundance can correlate with an insect cell line that is highly productive of viral vectors).

[0196] Figure 1 shows a schematic diagram of an exemplary technique for screening insect cells for viral vector expression or other viral vector characteristics. The provided technique uses an insect cell library modified to contain an identifier (e.g., including a barcode and / or library variant) along with appropriate genetic elements for packaging the identifier into a viral vector (e.g., viral repeat sequences, e.g., AAV ITRs for expression of an AAV vector) (shown in Figure 1, step A). ​​In some embodiments, the identifier is present on a library construct, which can be a single contiguous polynucleotide or two or more discontinuous polynucleotides. In some embodiments, the library construct further comprises, for example, at least one library variant. In some embodiments, the library variant can affect viral vector production or other properties. In some embodiments, the library variant can cause a perturbation, e.g., one or more genetic modifications that can affect viral vector production or other characteristics. In some embodiments, insect cells are genetically modified to contain one or more modified sequences that include an identifier located between the viral packaging sequence and, optionally, between any of the perturbation, library variant, payload, perturbed accessory sequence, trans-acting integration sequence, and / or cis-acting integration sequence.

[0197] The insect cells of the insect cell library may also contain sufficient genetic material to express the viral vector (e.g., already present as an episome and / or integrated into the insect cell genome) or may be modified (e.g., transfected) to contain the genetic material (shown in Figure 1, step B). Transfection or transduction with the genetic material (e.g., one or more modified sequences) of the insect cell library may be used to generate a viral vector-producing insect cell library.

[0198] The genetic material can include, for example, viral genes for replication and viral genes encoding necessary viral proteins, such as capsid proteins. In the case of AAV vectors, sufficient genetic material to express the viral vector includes (i) genes encoding AAV Rep and AAV Cap proteins, and (ii) adenoviral genes necessary to support AAV replication (e.g., E2, E4, and VA RNA). In some embodiments, the viral vector further comprises a payload (e.g., a nucleic acid encoding the payload). In some embodiments, the payload comprises an expression cassette having a promoter, an ORF, and a polyA signal. In some embodiments, insect cells of the insect cell library are transfected with sufficient genetic material to express the viral vector comprising the payload and the identifier.

[0199] In some embodiments, the provided technology involves a unique approach in which the viral vector incorporates an identifier (e.g., in the viral vector genome, e.g., between viral repeat sequences). Thus, each clonal population of insect cells in the library expresses a viral vector with a unique identifier. This allows for direct characterization of the viral vector and identification of the insect cell that produced it. Generally, the viral vector-producing insect cell library of the present disclosure contains at least one modified sequence (e.g., a viral repeat sequence, e.g., an AAV ITR for expression of an AAV vector) containing an identifier located between sequences for packaging the identifier into the viral vector, and sufficient genetic material for expression of the viral vector of interest. The insect cells in the viral vector-producing insect cell library can also contain one or more (e.g., up to 100 unique) library variants and / or perturbations. One or more perturbations can be present in the insect cells and / or the viral vectors expressed by the insect cells.

[0200] Any of the modified sequences (e.g., encoding the viral vector, identifier, and / or other modified sequences) can be present episomally (e.g., on one or more plasmids) and / or integrated into the genome of the insect cell. For example, in some embodiments, one or more nucleic acid sequences essential for the production of a viral vector can be integrated into an insect cell. In some embodiments, one or more nucleic acid sequences essential for the production of a viral vector can be present episomally in an insect cell. In some embodiments, one or more nucleic acid sequences essential for the production of a viral vector can be inducibly expressed (e.g., under the control of an inducible promoter).

[0201] In some embodiments, the viral vector-producing insect cell library is cultured and the viral vectors are recovered using any suitable method(s) known in the art (shown in Figure 1, step C). In some embodiments, all viral vectors produced by the insect cells of the library are recovered. In some embodiments, the viral vectors produced by the insect cells of the library are recovered according to time intervals. For example, viral vectors can be recovered daily, every two days, every three days, or more frequently to assess viral vector production over a period of time. In some embodiments, the insect cells can be washed and the viral vectors recovered after an extended period of time (e.g., to assess sustained viral vector production). In some embodiments, the genetic material of the viral vector (e.g., including the identifier) ​​is isolated.

[0202] The viral vectors can be pooled and the genetic material sequenced (e.g., using next-generation sequencing) (shown in Figure 1, step D). The presence of the identifier in the pool of viral vectors indicates that the insect cells or clonal insect cell lines (which also have the identifier) ​​in the insect cell library are capable of producing viral vectors. In some embodiments, the relative abundance of one or more identifiers is measured. In some embodiments, the relative abundance of the identifier measured in the pool of viral vectors correlates with the relative viral vector production by the corresponding cell lines. In some embodiments, the relative abundance of the identifier can be used to determine the amount (e.g., relative quantity) of a particular viral vector produced by different insect cells in the library. In some embodiments, the abundance of the identifier in the viral vector pool can be used to determine the effect of various modified sequences (e.g., library variants and / or perturbations) on the ability of the insect cells in the library to produce viral vectors.

[0203] The present disclosure recognizes that the amount or abundance of viral vectors may reflect bias (e.g., cell division or increased cell number of a particular insect cell or insect clonal cell line) and not necessarily reflect the viral vector production level associated with the insect cell or clonal cell line. Thus, in some embodiments, the amount or abundance of viral vectors may be normalized. In some embodiments, the amount or abundance of detected viral vectors may be normalized to, for example, the amount of cells and / or the amount of cellular DNA. In some embodiments, a cell library contains approximately the same number of cells within each sample of cells in the library.

[0204] Insect cells associated with desired viral vector characteristics (e.g., high expression) can be identified, and their modified sequences can be determined (e.g., by genome sequencing). For example, insect cells can be selected that exhibit increased viral vector production under good manufacturing practice (e.g., under current Good Manufacturing Practice (cGMP)) compared to reference cells. Increased viral vector production can be an increase in the number of viral vectors over a period of time or production over a longer period of time compared to the reference cells. In some embodiments, insect cells that produce viral vectors for a longer period of time compared to the reference cells can be selected (e.g., by increased viability, increased genomic stability, and / or increased duration of viral vector production). Reference cells can include comparable insect cells that do not contain the modified sequence and / or standard cells (e.g., cells capable of producing viral vectors).

[0205] Selected insect cell candidates can generally be used to produce viral vector(s), and / or the identified corresponding library variants and / or perturbations can be used to inform the construction of novel insect cell libraries (shown in Figure 1, panel F). The library-based platform approach can be iteratively repeated until modified insect cells with desired characteristics and / or expressing viral vectors at desired quantities are identified. Modified sequences associated with desired properties can be analyzed, for example, by developing machine learning (ML) models using machine learning approaches. The trained machine learning models can inform future design and help reduce the number of insect cell libraries to be screened and / or the size of each insect cell library, thereby reducing time and costs. In some embodiments, insect cell libraries can be designed and / or methods can be performed to identify modified sequences (e.g., combinations of two or more modified sequences) that interact synergistically in insect cells to have desired properties (e.g., a particular level of viral vector production). In some embodiments, the resulting insect cells obtained from the platform technologies described herein can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more perturbations). In some embodiments, the provided methods are useful for producing, screening, selecting, modifying, and / or identifying insect cells with desired characteristics for viral vector production (e.g., production at a certain level, production for a desired period of time, etc.). In some embodiments, the provided methods are useful for producing, screening, selecting, modifying, and / or identifying one or more perturbations or combinations of perturbations that affect viral vector production. In some embodiments, the selected candidate insect cells can be used for producing viral vector(s) after additional modification (e.g., removal of identifiers).In some embodiments, the identified corresponding library variants and / or perturbations can be used to inform the construction of similar insect cell lines or cell lines (e.g., perturbations can be introduced into the same ancestral cell line, a similar ancestral cell line, where one or more polynucleotides comprising one or more nucleic acid sequences essential for viral vector production are integrated into the insect cell genome, and / or a very different ancestral cell line).

[0206] In some embodiments, insect cells are modified to contain one or more components of a viral vector under the control of an inducible transcriptional control element (e.g., a promoter). In some embodiments, insect cells containing genetic elements of a viral vector are engineered to further contain a library construct. In some embodiments, insect cells are modified to contain a polynucleotide (e.g., episomally and / or integrated into the insect genome) having one or more elements essential for production of a viral vector under the control of an inducible transcriptional control element, and further contain a library construct.

[0207] Those skilled in the art will recognize that while the schematic diagram in FIG. 1 illustrates modifying an insect cell library to include one or more modified sequences and one or more identifiers, followed by introducing genetic elements of a viral vector, these steps for modifying an insect cell library to express a viral vector can be performed in any order using any method known in the art. For example, modifying a viral vector to express an insect cell library can be performed in any order using any method known in the art. For example, in some embodiments, the modified sequence(s), identifier(s), and viral vector elements are introduced simultaneously or substantially simultaneously. In some embodiments, an insect cell library is generated containing unique modified sequences and polynucleotides essential for forming a viral vector, followed by introducing the identifier. In some embodiments, an insect cell library is generated containing unique modified sequences including the identifier, followed by introducing the polynucleotides essential for forming a viral vector.

[0208] The provided methods can be used to generate, identify, and / or select insect cells with desired characteristics for the production of any desired viral vector. Generally, such viral vectors are functional for their desired purpose. For example, in some embodiments, viral vectors for gene therapy are functional to deliver a payload (e.g., a nucleic acid to a target cell). In some embodiments, the viral vector is an oncolytic viral vector capable of killing cancer cells. In some embodiments, the viral vector is therapeutically active. However, in some embodiments, the provided methods may result in a non-functional viral vector that lacks one or more functional characteristics but retains other characteristics of interest. In some embodiments, the viral vector is non-functional or has reduced functionality for a particular characteristic. For example, in some embodiments, the viral vector may have reduced ability or may be unable to deliver a payload. In some embodiments, the viral vector may have reduced ability to kill cancer cells. In some embodiments, the viral vector may be therapeutically inactive.

[0209] viral vectors In some embodiments, the insect cell lines of the present disclosure are useful for producing viral vectors. In some embodiments, the produced viral vectors are used as biologics and / or therapeutics themselves. In some embodiments, the produced viral vectors are used in research, production, and / or manufacturing processes that generate many biologics and / or therapeutics. For example, viral vectors can be used in many ways, including, but not limited to, vaccines, cancer therapy (e.g., oncolytic therapy), and / or gene therapy (e.g., in vivo gene and / or genome editing). As another example, viral vectors can be used in many ways, including, but not limited to, research, production, and / or manufacturing of vaccines, cancer therapy (e.g., oncolytic therapy), gene therapy (e.g., ex vivo gene and / or genome editing), and / or cell therapy (e.g., ex vivo gene and / or genome editing). Thus, there is a wide range of viral vectors for these various uses.

[0210] The methods of the present disclosure can be used to select and / or identify insect cells that have properties beneficial for the expression of a viral vector, for example, any viral vector known in the art or described herein, and the present disclosure is not limited to a particular viral vector.

[0211] Viral vectors can have different sizes, carrying capacities, and different genomic structures (e.g., DNA and / or RNA, and single-stranded and / or double-stranded). In some embodiments, viral vectors can be used to deliver nucleic acids to cells for transient or long-term expression. In some embodiments, viral vectors have a broad range of host cells. In some embodiments, viral vectors can have limited and / or specific host cell types.

[0212] The insect cells and / or populations of insect cells provided comprise one or more polynucleotides comprising one or more nucleic acid sequences essential for viral vector production. In some embodiments, the polynucleotide comprising one or more nucleic acid sequences essential for viral vector production is present in the insect cell as an episome. In some embodiments, the polynucleotide comprising one or more nucleic acid sequences essential for viral vector production is present in the insect cell genome. In some embodiments, the one or more nucleic acid sequences essential for viral vector production are under the control of an inducible transcriptional control element. For example, in some specific embodiments, the one or more nucleic acid sequences essential for viral vector production can be integrated into the insect cell genome under the control of an inducible transcriptional control element (e.g., an inducible promoter and / or an inducible enhancer). Viruses from which viral vectors are derived include adeno-associated virus (AAV), adenovirus, baculovirus (e.g., Autographa californica nucleopolyhedrovirus (AcMNPV)), lentivirus, alphavirus (e.g., Sindbis virus), retrovirus (e.g., gammaretrovirus), polyomavirus (e.g., Simian virus 40 (SV40)), papillomavirus (e.g., bovine papillomavirus (BPV)), poxvirus (e.g., vaccinia virus), herpes simplex virus (HSV), measles virus, rhabdovirus (e.g., rabies virus), vesicular stomatitis virus (VSV), picornavirus (e.g., poxvirus, These include, but are not limited to, viruses derived from viruses such as lyoviruses, reoviruses, senecaviruses, echoviruses (e.g., RIGVIR), Semliki Forest virus (SFV), flaviviruses, anelloviruses (https: / / www.ringtx.com), Newcastle disease virus (NDV), paramyxoviruses (e.g., Sendai virus), Sendai virus vectors, orthomyxoviruses (e.g., influenza virus), coronaviruses, and hybrid and / or modified viruses and / or viral vectors.

[0213] In certain embodiments, with respect to the present disclosure, the viral vector is derived from an adeno-associated virus, an adenovirus, a baculovirus, a lentivirus, a retrovirus, and / or a herpes simplex virus.

[0214] In some embodiments, the insect cell lines of the present disclosure are useful for producing viral vectors, such as adeno-associated viral vectors, adenoviral vectors, baculoviral vectors, lentiviral vectors, retroviral vectors, and / or herpes simplex viral vectors.

[0215] In some embodiments, the viral vectors produced of the present disclosure are suitable for production and / or manufacturing processes using current Good Manufacturing Practice (cGMP) to produce many biologics and / or therapeutics. In some embodiments, the viral vectors are suitable for use in industrial-scale manufacturing of biologics. In some embodiments, the viral vectors are suitable for use in manufacturing methods that comply with national regulatory standards (e.g., FDA and / or EMA regulatory standards).

[0216] The viral vector may be live and attenuated. In some embodiments, the viral vector may be replication-conditional. In some embodiments, the viral vector may be replication-defective. In some embodiments, the viral vector may be replication-incompetent. In some embodiments, the viral vector is replication-defective. In some embodiments, the viral vector is replication-competent. In some embodiments, the viral vector is non-pathogenic.

[0217] In some embodiments, the present disclosure provides a unique approach in which a viral vector takes up an identifier from an insect cell that expresses it and incorporates it into its viral nucleic acid (e.g., a viral genome or construct, e.g., between viral repeat sequences). Thus, insect cells and / or viral vectors produced by the disclosed technology have an identifier within their viral nucleic acid (e.g., a viral genome or construct, e.g., between viral repeat sequences). In some embodiments, viral vectors produced by the disclosed insect cells and / or methods comprise modified sequences, such as a capsid and a nucleic acid, where the nucleic acid comprises (i) a payload, (ii) an identifier, and (iii) one or more sequences of the viral genome (e.g., for AAV, this may be between the AAV ITR sequence(s)). In some embodiments, the identifier and / or payload, or portions thereof, are subsequently removed from the viral vector. In some embodiments, the identifier and / or payload, or portions thereof, are subsequently removed from the viral vector, and the payload, or portions thereof, are replaced with another payload, or portions thereof.

[0218] Adeno-associated virus (AAV) vectors In some embodiments, the viral vector produced by the methods and insect cells of the present disclosure is an adeno-associated viral (AAV) vector. AAV is a viral vector commonly used for gene delivery. In some embodiments, AAV vectors have low immunogenicity (e.g., in humans). In some embodiments, AAV vectors are compatible with a wide range of host cells. In some embodiments, AAV vectors can transduce both dividing and quiescent cells. In some embodiments, the insect cells of the present disclosure produce the AAV vectors described herein.

[0219] In some embodiments, the present disclosure provides nucleic acid sequences encoding one or more elements essential for AAV vector production. Essential elements of an AAV vector may include Rep proteins and / or capsid (Cap) proteins (e.g., VP1, VP2, and VP3, which form the AAV capsid). In some embodiments, essential elements of an AAV vector may be encoded on one or more constructs (e.g., which may be integrated or present as an episome in an insect cell). In some embodiments, nucleic acids encoding one or more elements essential for AAV vector production are integrated into the genome of an insect cell. In some embodiments, nucleic acids encoding one or more elements essential for AAV vector production are present as an episome in an insect cell.

[0220] In some embodiments, the present disclosure provides an AAV vector comprising a capsid containing a payload and a nucleic acid, hi some embodiments, the AAV vector has an icosahedral protein capsid that encapsulates a linear, single-stranded DNA nucleic acid.

[0221] In some embodiments, the viral vectors produced by the insect cells and / or methods of the present disclosure comprise an AAV capsid and nucleic acid, wherein the nucleic acid comprises (i) a payload, (ii) an identifier (e.g., comprising a barcode and / or library variant), and (iii) two ITR sequences (e.g., from an AAV).

[0222] In some embodiments, the AAV vector is derived from human AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or derivatives thereof. In some embodiments, the AAV vector is a synthetic and / or hybrid human AAV vector. In some embodiments, the AAV vector is derived from bovine AAV (b-AAV), canine AAV (CAAV), mouse AAV1, goat AAV, rat AAV, or avian AAV (AAAV).

[0223] In some embodiments, an AAV vector may be described as having a serotype, which is a description of the strain of capsid and the strain of specific sequences of nucleic acid (e.g., ITRs). For example, in some embodiments, an AAV vector may be described as AAV2, in which case the vector has an AAV2 capsid and nucleic acid containing characteristic AAV2 inverted terminal repeat sequences (ITRs). In some embodiments, an AAV vector may be described as pseudotyped, in which case the capsid and ITRs are derived from different AAV strains; for example, AAV2 / 9 refers to an AAV vector comprising a construct utilizing AAV2 ITRs and an AAV9 capsid.

[0224] In some embodiments, the AAV vector has no serotype and / or pseudotype, hi some embodiments, the AAV vector comprises a modified AAV capsid and / or ITRs (e.g., no significant homology to those of known AAV serotypes).

[0225] In some embodiments, the AAV vector of the present disclosure comprises an AAV capsid. In some embodiments, the AAV capsid is derived from or derived from an AAV capsid of an AAV2, 3, 4, 5, 6, 7, 8, 9, 10, rh8, rh10, rh39, rh43, or Anc80 serotype, or is a hybrid of one or more thereof. In some embodiments, the AAV capsid is derived from an AAV ancestral serotype. In some embodiments, the AAV capsid is an ancestral (Anc) AAV capsid. Anc capsids are generated from the sequence of a construct constructed using evolutionary probability and evolutionary modeling to determine a predicted ancestral sequence. Therefore, it is unknown whether the Anc capsid / construct sequence exists in nature. In some embodiments, the AAV capsid is an artificially modified sequence (e.g., does not have significant homology to known AAV serotype capsids).

[0226] As provided herein, the AAV vectors of the disclosure can comprise any combination of AAV capsid and AAV nucleic acid (e.g., including a payload and / or AAV ITRs), such as a wild-type or variant AAV capsid that encapsidates an AAV nucleic acid including an identifier and / or payload flanked by AAV-derived ITRs.

[0227] Generally, AAV nucleic acids consist of single-stranded deoxyribonucleic acid (ssDNA). In some embodiments, AAV nucleic acids contain one or more components derived from or modified from a natural AAV genome. In some embodiments, AAV nucleic acids contain inverted terminal repeats (ITRs) derived from or modified from AAV. In some embodiments, AAV nucleic acids contain a payload sequence and two ITRs. In some embodiments, AAV vectors contain a capsid and a payload sequence, and two viral repeat sequences, e.g., an ssDNA containing an ITR sequence, one at each end (5' and 3') of the DNA strand.

[0228] In some embodiments, the provided AAV nucleic acid comprises a coding sequence and one or more regulatory and / or control sequences, and optionally a payload comprising AAV-derived inverted terminal repeats (ITRs) at the 5' and 3' positions. In some embodiments, the provided AAV nucleic acid is packaged into an AAV capsid to form an AAV vector.

[0229] In certain embodiments, the viral vector comprises a nucleic acid comprising an identifier and / or payload sequence and associated regulatory elements flanked by 5' or "left" and 3' or "right" AAV ITR sequences. The 5' and left designations refer to the position of the ITR sequences relative to the overall construct and are read from left to right in the sense orientation. One of skill in the art will understand how to modify a given ITR sequence for use as either a 5' / left or 3' / right ITR, or an antisense version thereof.

[0230] In some embodiments, the AAV nucleic acid of the AAV vectors described herein generally comprises cis-acting 5' and 3' ITR sequences (see, e.g., B.J. Carter, in "Handbook of Parvoviruses," ed., P. Tijsser, CRC Press, pp. 155-168, 1990, which is incorporated herein by reference in its entirety). In some embodiments, at least 80% (e.g., at least 85%, at least 90%, or at least 95%) of the common ITR sequences are incorporated into the constructs provided herein. The ability to modify these ITR sequences is within the skill of one in the art (see, e.g., Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory, New York, 1989; and K. Fisher et al., J. See, e.g., Virol. 70:520-532, 1996, each of which is incorporated by reference in its entirety. In some embodiments, the identifier and / or payload sequence are flanked by 5' and 3' AAV ITR sequences. In some embodiments, the AAV nucleic acid comprises an identifier and a payload flanked by 5' and 3' AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including currently identified AAV types.

[0231] In some embodiments, the nucleic acid of the AAV vector comprises a payload, an identifier, and two AAV ITRs. In some embodiments, the AAV vector comprises a capsid and (i) a payload and / or identifier, and (ii) a dsDNA comprising two AAV ITR sequences, one at each end (5' and 3') of the DNA strand.

[0232] Generally, ITRs can form hairpins. The ability to form hairpins contributes to the self-priming ability of ITRs, allowing for primase-independent synthesis of the second DNA strand. ITRs also aid in the efficient encapsulation of AAV constructs in AAV vectors. AAV ITR sequences can be obtained from any known AAV, including insect AAV types.

[0233] In some embodiments, the ITRs contain one or more modifications of the native ITR sequence, such as truncations, deletions, substitutions, or insertions. The ability to modify these ITR sequences is within the skill of those in the art (see, e.g., 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), each of which is incorporated herein by reference in its entirety). For example, the ITR sequence from AAV2 is approximately 145 nucleotides in length. In some embodiments, the ITRs contain fewer than 145 nucleotides, e.g., 127, 130, 134, or 141 nucleotides. For example, in some embodiments, the ITR comprises 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, or 145 nucleotides.

[0234] In some embodiments, the AAV vector payload also comprises conventional control elements operably linked to the coding sequence in a manner that allows for its transcription, translation, and / or expression in cells transfected by the construct or infected with a viral vector produced by the present disclosure. In some embodiments, the AAV vector payload optionally comprises a promoter, an enhancer, an untranslated region (e.g., 5'UTR, 3'UTR), a Kozak sequence, an internal ribosome entry site (IRES), a splicing site (e.g., acceptor site, donor site), a polyadenylation site, and / or any combination thereof.

[0235] In some embodiments, the AAV vector payload is less than 4kb. In some embodiments, the AAV vector payload can comprise a sequence that is at least 500bp, at least 1kb, at least 1.5kb, at least 2kb, at least 2.5kb, at least 3kb, at least 3.5kb, at least 4kb, or at least 4.5kb. In some embodiments, the AAV vector payload can comprise a sequence that is at most 7.5kb, at most 7kb, at most 6.5kb, at most 6kb, at most 5.5kb, at most 5kb, at most 4.5kb, at most 4kb, at most 3.5kb, at most 3kb, or at most 2.5kb. In some embodiments, the AAV vector payload may comprise a sequence of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, or about 4 kb to about 5 kb.

[0236] In some embodiments, the AAV vector is capable of directing long-term expression of a payload. In some embodiments, the AAV vector is capable of directing transient expression of a payload. In some embodiments, the AAV vector produced by the insect cells and / or methods of the present disclosure comprises an AAV capsid and a nucleic acid, the nucleic acid comprising (i) a payload, (ii) an identifier (e.g., a barcode and / or library variant), and (iii) two viral repeat sequences (e.g., AAV-derived ITRs).

[0237] In some embodiments, insect cells in the insect cell library are genetically modified to contain one or more nucleic acid sequences essential for AAV vector production. In some embodiments, insect cells in the insect cell library may have one or more AAV vector components provided, such as rep sequences, cap sequences, and helper functions, necessary for AAV vector production. In some embodiments, one or more components of the AAV vector (e.g., the AAV Rep gene, the AAV Cap gene, one or more helper genes, or a combination thereof) are under the control of an inducible transcriptional control element (e.g., a promoter and / or enhancer). In some embodiments, insect cells (e.g., in a population of insect cells) contain (i) an identifier disposed between two viral repeat sequences, and (ii) one or more polynucleotides comprising an AAV Rep gene, an AAV Cap gene, one or more helper genes, and / or a combination thereof, whereupon the insect cell(s) produce an AAV vector comprising the identifier. In some embodiments, the AAV vector comprises a payload. In some embodiments, the identifier and / or payload, or portions thereof, are subsequently removed from the AAV vector. In some embodiments where the identifier and / or payload or portion thereof is removed from the AAV vector, the payload or portion thereof is replaced with a different payload or portion thereof.

[0238] Adenovirus vectors In some embodiments, the viral vector produced by the methods and insect cells of the present disclosure is an adenovirus vector. Adenoviruses are non-enveloped viruses that are commonly used as vaccines due to the strong immunogenic response they induce. Some adenoviruses have been utilized in cancer therapy due to their ability to preferentially infect and kill cancer cells.

[0239] In some embodiments, the adenoviral vector is derived from a human adenovirus. Human adenoviruses encompass a family of at least 51 serotypes, divided into several subgroups. For example, subgroup A includes adenovirus serotypes 12, 18, and 31. Subgroup B includes adenovirus serotypes 3, 7, 11a, 11p, 14, 16, 21, 34, 35, and 50. Subgroup C includes adenovirus serotypes 1, 2, 5, and 6. Subgroup D includes adenovirus serotypes 8, 9, 10, 13, 15, 17, 19, 19p, 20, 22-30, 32, 33, 36-39, 42-49, and 51. Subgroup E includes adenovirus serotype 4. Subgroup F includes adenovirus serotypes 40 and 41. The adenoviral vectors of the present disclosure can be of any adenovirus group, subgroup, and / or serotype.

[0240] In some embodiments, the adenoviral vector is derived from any serotype, such as serotype 1 through serotype 51 (e.g., 1, 2, 4, 5, 51). For example, in some embodiments, the adenovirus is adenovirus type 2 or adenovirus type 5. In some specific embodiments, the adenoviral vector is derived at least in part from adenovirus type 5.

[0241] In some embodiments, the adenoviral vector is replication-deficient (e.g., certain essential viral genes have been deleted and / or replaced with payload sequences). Any of the adenoviral vectors used in the methods described herein can contain a deletion in any one or more of the E1, E2a, E2b, E3, or E4 coding regions. In some embodiments, the adenoviral vector is replication-deficient and lacks the E4 locus (e.g., the E4 coding region is deleted). In some embodiments, replication-deficient adenoviral vectors are useful as vaccines for cancer therapy and / or gene therapy.

[0242] Generally, adenoviruses are characterized by high transduction efficiency and direct high transgene expression. In some embodiments, adenoviral vectors can direct transient expression of a payload. In some embodiments, adenoviral vectors direct transient expression of a payload in target cells.

[0243] In some embodiments, the adenoviral vector is replication-competent. For example, a replication-competent adenoviral vector (e.g., an oncolytic vector) can be engineered to replicate preferentially in cancer cells and destroy them through the natural lytic viral replication process.

[0244] In some embodiments, the present disclosure provides an adenoviral vector comprising a capsid containing a payload and a nucleic acid. In some embodiments, the adenoviral vector has an icosahedral protein capsid that encapsulates a linear double-stranded nucleic acid. In some embodiments, the adenoviral vector is approximately 90-100 nm in diameter. In some embodiments, the adenoviral vector has an icosahedral protein capsid that encapsulates a linear double-stranded DNA.

[0245] Generally, the nucleic acid of an adenoviral vector consists of double-stranded DNA (dsDNA) and is derived from or has one or more modified components of the native adenoviral genome. In some embodiments, the nucleic acid of an adenoviral vector is derived from an adenovirus of any serotype (e.g., adenovirus type 5) or contains modified inverted terminal repeats (ITRs). In some embodiments, the nucleic acid of an adenoviral vector contains a payload and two viral repeat sequences, e.g., ITRs. In some embodiments, the nucleic acid of an adenoviral vector contains an identifier and two viral repeat sequences, e.g., ITRs. In some embodiments, the nucleic acid of an adenoviral vector contains a payload, an identifier, and two viral repeat sequences, e.g., ITRs. In some embodiments, the adenoviral vector contains a capsid and (i) a payload and / or identifier, and (ii) dsDNA containing two ITR sequences, one at each end (5' and 3') of the DNA strand.

[0246] In some embodiments, the adenoviral ITRs have sequences with a length of about 40 bp to about 200 bp. For example, the ITR of human adenovirus type 5 is 103 bp. In some embodiments, the adenoviral ITRs include lengths of about 30 bp, about 40 bp, about 50 bp, about 60 bp, about 70 bp, about 80 bp, about 90 bp, about 100 bp, about 110 bp, about 120 bp, about 130 bp, about 140 bp, about 150 bp, about 160 bp, about 170 bp, about 180 bp, about 190 bp, about 200 bp, about 220 bp, about 240 bp, about 260 bp, about 280 bp, about 300 bp, about 325 bp, about 350 bp, about 375 bp, or about 400 bp. In some specific embodiments, the adenoviral ITR sequences are about 50 to about 250 bp.

[0247] In some embodiments, provided adenoviral vectors comprise an adenovirus-derived capsid and a nucleic acid comprising a payload and one or more adenovirus-derived sequences (e.g., ITRs). In some embodiments, provided adenoviral vectors comprise an adenovirus-derived capsid and a nucleic acid comprising an identifier, a payload, and at least two viral repeat sequences. In some embodiments, the adenoviral vector comprises an identifier comprising a barcode and / or a library variant. In some embodiments, the adenoviral vector payload comprises a coding sequence and one or more regulatory and / or control sequences. In some embodiments, the adenoviral vector comprises 5' and 3' adenovirus-derived inverted terminal repeats (ITRs). In some specific embodiments, the adenoviral vector comprises a capsid and a modified adenoviral genome, where the modified genome comprises a deletion of specific sequences (e.g., an E1 deletion and / or an E3 deletion) and an insertion of a payload and / or an identifier.

[0248] In some embodiments, the payload of the adenoviral vector is less than 7.5 kb. In some embodiments, the payload of the adenoviral vector may comprise a sequence that is at least 500 bp, at least 1 kb, at least 1.5 kb, at least 2 kb, at least 2.5 kb, at least 3 kb, at least 3.5 kb, at least 4 kb, at least 4.5 kb, at least 5 kb, at least 5.5 kb, at least 6 kb, at least 6.5 kb, at least 7 kb, or at least 7.5 kb. In some embodiments, the payload of the adenoviral vector may comprise a sequence that is at most 10 kb, at most 9.5 kb, at most 9 kb, at most 8.5 kb, at most 8 kb, at most 7.5 kb, at most 7 kb, at most 6.5 kb, at most 6 kb, at most 5.5 kb, at most 5 kb, at most 4.5 kb, or at most 4 kb. In some embodiments, the payload of the adenoviral vector may comprise a sequence that is about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 10 kb, about 2 kb to about 4 kb, about 2 kb to about 6 kb, about 2 kb to about 8 kb, about 2 kb to about 10 kb, about 4 kb to about 6 kb, about 4 kb to about 8 kb, or about 5 kb to about 8 kb.

[0249] In some embodiments, the adenoviral vector produced by the insect cells and / or methods of the present disclosure comprises an adenoviral capsid and nucleic acid, wherein the nucleic acid comprises (i) a payload, (ii) an identifier (e.g., a barcode and / or library variant), and (iii) two viral repeat sequences (e.g., adenovirus-derived ITRs).

[0250] In some embodiments, insect cells in the insect cell library are genetically modified to contain one or more nucleic acid sequences essential for the production of adenoviral vectors. In some embodiments, insect cells in the insect cell library can have one or more adenoviral vector components provided, such as a rep sequence, a cap sequence, and helper functions, necessary for producing adenoviral vectors. In some embodiments, one or more components of the adenoviral vector (e.g., an adenoviral Rep gene, an adenoviral Cap gene, one or more helper genes, or a combination thereof) are under the control of an inducible transcriptional control element (e.g., a promoter and / or enhancer). In some embodiments, insect cells (e.g., in a population of insect cells) contain (i) an identifier disposed between two viral repeat sequences, and (ii) one or more polynucleotides comprising an adenoviral Rep gene, an adenoviral Cap gene, one or more helper genes, and / or a combination thereof, where the insect cell(s) produce adenoviral vectors comprising the identifier. In some embodiments, the adenoviral vector comprises a payload. In some embodiments, the identifier and / or payload, or portion thereof, is subsequently removed from the adenoviral vector. In some embodiments where the identifier and / or payload, or portion thereof, is removed from the adenoviral vector, the payload, or portion thereof, is replaced with a different payload, or portion thereof.

[0251] Retroviral and lentiviral vectors In some embodiments, the viral vectors produced by the methods and insect cells of the present disclosure are retroviral vectors. Retroviruses are enveloped viruses that replicate in host cells by transcribing their RNA into DNA using the viral reverse transcriptase. Retroviral DNA replicates as part of the host genome and is called a provirus. Retroviral vectors can be based on or derived from murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), ecotropic retroviruses, simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof, but are not limited to these.

[0252] In some embodiments, the viral vector produced by the disclosed methods and insect cells is a lentiviral vector. Lentiviral vectors are versatile tools due to their ability to transduce non-dividing cells. In some embodiments, lentiviral vectors can infect both dividing and non-dividing cells. Generally, lentiviral vectors allow for long-term and / or stable gene expression and are integrated into the host cell genome.

[0253] Lentiviruses are enveloped particles with a diameter of about 80 to about 120 nm. In some embodiments, the present disclosure provides lentiviral vectors having a diameter within the range of about 50 nm to about 200 nm. In some embodiments, the present disclosure provides lentiviral vectors having a diameter within the range of about 80 nm to about 120 nm.

[0254] Lentiviruses can include several structural proteins, including a matrix, capsid, nucleocapsid, envelope, and reverse transcriptase protein. In some embodiments, the present disclosure provides a lentiviral vector comprising a capsid and an envelope. In some embodiments, the present disclosure provides a lentiviral vector comprising a capsid, an envelope, and a nucleic acid. In some embodiments, the present disclosure provides a lentiviral vector comprising a capsid, an envelope, and a nucleic acid comprising a payload and / or identifier.

[0255] In some embodiments, the lentiviral vector produced by the insect cells and / or methods of the present disclosure comprises a lentiviral capsid (or a derivative thereof), an envelope, and a nucleic acid, wherein the nucleic acid comprises (i) a payload, (ii) an identifier, and (iii) two long terminal repeat (LTR) sequences (e.g., derived from a lentivirus). In some embodiments, the two LTR sequences are capable of packaging the nucleic acid into the lentiviral vector. In some embodiments, the lentiviral vector comprises a lentiviral Psi sequence (or a derivative or modified variant thereof).

[0256] In some embodiments, lentiviral vectors include vectors based on human immunodeficiency virus (HIV-1), HIV-2, feline immunodeficiency virus (FIV), equine infectious anemia virus, simian immunodeficiency virus (SIV), and Maedi-Visna virus (MVV). In some embodiments, the lentiviral nucleic acid (i.e., the modified genome) and envelope glycoprotein are based on different viruses, such that the resulting viral vector is pseudotyped.

[0257] In some embodiments, the lentiviral vector is derived from HIV-1. In some embodiments, the lentiviral vector is derived from HIV-1 and comprises a capsid protein and a nucleic acid comprising (i) a payload, (ii) an identifier, and (iii) two long terminal repeat (LTR) sequences (e.g., derived from a lentivirus). In some embodiments, the lentiviral vector is an HIV vector, and the insect cell comprises two viral repeat sequences comprising HIV LTR sequences. In some embodiments, the lentiviral vector is an SIV vector, and the insect cell comprises two viral repeat sequences comprising SIV LTR sequences. In some embodiments, the lentiviral vector is an equine infectious anemia virus vector, and the insect cell comprises two viral repeat sequences comprising equine infectious anemia virus LTR sequences. In some embodiments, the lentiviral vector is an FIV vector, and the insect cell comprises two viral repeat sequences comprising FIV LTR sequences. In some embodiments, the lentiviral vector is a visna virus vector, and the insect cell comprises two viral repeat sequences comprising visna virus LTR sequences.

[0258] In some embodiments, the lentiviral vector comprises a gag protein or a fragment thereof. In some embodiments, the lentiviral vector comprises a gag protein comprising one or more domains selected from the matrix (MA), capsid (CA), and nucleocapsid (NC) domains. In some embodiments, the lentiviral vector comprises an envelope protein or a fragment thereof. In some embodiments, the lentiviral vector is a pseudotyped lentiviral vector, wherein the gag protein and the envelope protein are derived from different viruses. In some embodiments, the lentiviral vector comprises a gag protein and / or an env protein derived from a human immunodeficiency virus (HIV) vector, a simian immunodeficiency virus (SIV) vector, an equine infectious anemia virus vector, a feline immunodeficiency virus vector, a visna virus vector, or derivatives thereof.

[0259] In some embodiments, the lentiviral vector comprises (i) a lentiviral gag gene, (ii) a lentiviral env gene, (iii) a lentiviral pol gene, or (iv) a combination thereof. In some embodiments, the insect cells expressing the lentiviral vector comprise one or more of (i) a lentiviral gag gene, (ii) a lentiviral env gene, and (iii) a lentiviral pol gene.

[0260] In some embodiments, safety features are incorporated into lentiviral vectors, which may include, for example, self-inactivating long terminal repeats (LTRs) and an integration defect. In certain embodiments, the integration defect may be conferred by elements of the vector genome, but may also result from elements of the packaging system (e.g., a non-functional integrase protein that is not part of the vector genome but may be supplied in trans).

[0261] Lentiviruses have a single-stranded RNA (ssRNA) genome. In some embodiments, lentiviral vectors comprise a nucleic acid that is ssRNA and include a payload and sequences derived from a lentivirus, such as HIV-1 and / or SIV. In some embodiments, the payload is flanked by long terminal repeat (LTR) sequences. In some embodiments, lentiviral vectors comprise a nucleic acid that is ssRNA and include an identifier and viral repeat sequences (e.g., HIV-1 and / or SIV LTRs). In some embodiments, lentiviral vectors comprise a nucleic acid that is ssRNA and include a payload, an identifier, and viral repeat sequences (e.g., HIV-1 and / or SIV LTRs).

[0262] In some embodiments, the nucleic acid of the lentiviral vector may comprise sequences derived from the 5' and 3' LTRs of a lentivirus, particularly the R and U5 sequences derived from the 5' LTR and an inactivating or self-inactivating 3' LTR derived from a lentivirus. The LTR sequences may be LTR sequences derived from any lentivirus from any species. For example, they may be LTR sequences derived from HIV, SIV, FIV, or BIV. In some embodiments, the LTR sequences are HIV LTR sequences.

[0263] In some embodiments, the payload of the lentiviral vector is less than 8 kb. In some embodiments, the payload of the lentiviral vector can include a sequence that is at least 500 bp, at least 1 kb, at least 1.5 kb, at least 2 kb, at least 2.5 kb, at least 3 kb, at least 3.5 kb, at least 4 kb, at least 4.5 kb, at least 5 kb, at least 5.5 kb, at least 6 kb, at least 6.5 kb, at least 7 kb, at least 7.5 kb, or at least 8 kb. In some embodiments, the payload of the lentiviral vector can include a sequence that is at most 10 kb, at most 9.5 kb, at most 9 kb, at most 8.5 kb, at most 8 kb, at most 7.5 kb, at most 7 kb, at most 6.5 kb, at most 6 kb, at most 5.5 kb, at most 5 kb, at most 4.5 kb, or at most 4 kb. In some embodiments, the payload of the lentiviral vector may comprise a sequence that is about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 10 kb, about 2 kb to about 4 kb, about 2 kb to about 6 kb, about 2 kb to about 8 kb, about 2 kb to about 10 kb, about 4 kb to about 6 kb, about 4 kb to about 8 kb, or about 5 kb to about 8 kb.

[0264] In some embodiments, insect cells in the insect cell library are genetically modified to contain one or more nucleic acid sequences essential for the production of a lentiviral vector. In some embodiments, the insect cells in the insect cell library can have one or more lentiviral vector components provided. In some embodiments, one or more components of the lentiviral vector are under the control of an inducible transcriptional control element (e.g., a promoter and / or enhancer). In some embodiments, the insect cells (e.g., in a population of insect cells) contain (i) an identifier disposed between two viral repeat sequences and (ii) one or more polynucleotides essential for the production of a lentiviral vector, and the insect cell(s) produce a lentiviral vector containing the identifier. In some embodiments, the lentiviral vector contains a payload. In some embodiments, the identifier and / or payload, or a portion thereof, are subsequently removed from the lentiviral vector. In certain embodiments, where the identifier and / or payload, or a portion thereof, has been removed from the lentiviral vector, the payload, or a portion thereof, is replaced with a different payload, or a portion thereof.

[0265] HSV vector In some embodiments, the viral vector produced by the disclosed methods and insect cells is a herpes simplex virus (HSV) vector. HSV is a large enveloped virus with an icosahedral capsid containing a toroidal dsDNA genome. HSV is characterized in vivo by lifelong latent infection of neurons. This property makes HSV vectors useful for long-term expression of transgenes. In some embodiments, the HSV vector is a replication-competent attenuated vector, a replication-incompetent recombinant vector, or a replication-deficient helper-dependent vector.

[0266] In some embodiments, the HSV vector has a diameter within the range of about 120 nm to about 200 nm. In some embodiments, the HSV vector is an enveloped particle with a diameter of about 120 to about 200 nm. In some embodiments, the HSV vector has a diameter within the range of about 100 nm to about 200 nm.

[0267] In some embodiments, the HSV vector is derived from or is derived from herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), human cytomegalovirus (HCMV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human herpesvirus 6 and / or human herpesvirus 7, and / or derivatives thereof. In some embodiments, the HSV vector is derived from or is derived from HSV-1, HSV-2, or a combination thereof (e.g., comprises a capsid from HSV-1 and comprises nucleic acid sequences from HSV-2).

[0268] In some embodiments, the viral vector is an HSV-AAV hybrid vector.

[0269] The native HSV-1 genome consists of long and short unique segments (U L and U S ), which are each separated by an inverted repeat (TR) sequence. L / IR L and IR S / TR S ) adjacent to the ICP0, ICP4, ICP22, ICP27, and / or ICP47. In some embodiments, HSV vectors are modified to lack or delete ICP0, ICP4, ICP22, ICP27, and / or ICP47 to reduce toxicity. Methods and considerations for designing HSV vectors are known in the art, e.g., Manservigi, et al., Open Virol J. 2010;4:123-156.

[0270] In some embodiments, the disclosure provides an HSV vector comprising a nucleic acid comprising capsid proteins and a payload, hi some embodiments, the HSV vector comprises a capsid comprising VP5, VP19C, VP23, pre-VP22a, and / or mature protease (UL26 gene product).

[0271] In some embodiments, the HSV nucleic acid comprises a sequence obtained or derived from an HSV virus (e.g., TR L / IR L and / or IR S / TR S In some embodiments, the present disclosure provides a method for detecting a capsid protein comprising a capsid protein and an HSV viral repeat sequence (e.g., a TR repeat sequence). L / IR L and / or IR S / TR S In some embodiments, the present disclosure provides an HSV vector comprising a nucleic acid comprising a capsid, an envelope, and a payload. In some embodiments, the present disclosure provides an HSV vector comprising a nucleic acid comprising a capsid, an envelope, and a payload and an HSV viral repeat sequence (e.g., a TR L / IR L and / or IR S / TR S The present invention provides an HSV vector comprising a nucleic acid comprising the sequence

[0272] In some embodiments, the payload of the HSV vector is less than 100 kb. In some embodiments, the payload of the HSV vector can include a sequence that is at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 15 kb, at least 20 kb, at least 25 kb, at least 30 kb, at least 40 kb, or at least 50 kb. In some embodiments, the payload of the HSV vector may comprise a sequence that is up to 150kb, up to 140kb, up to 130kb, up to 120kb, up to 110kb, up to 100kb, up to 90kb, up to 80kb, up to 70kb, up to 60kb, up to 50kb, up to 40kb, up to 30kb, up to 25kb, up to 20kb, up to 15kb, or up to 10kb. In some embodiments, the payload of the lentiviral vector may comprise a sequence that is about 1 kb to about 150 kb, about 1 kb to about 100 kb, about 1 kb to about 50 kb, about 1 kb to about 25 kb, about 5 kb to about 100 kb, about 5 kb to about 90 kb, about 5 kb to about 80 kb, about 5 kb to about 70 kb, about 5 kb to about 60 kb, about 5 kb to about 50 kb, about 5 kb to about 40 kb, about 5 kb to about 30 kb, about 5 kb to about 25 kb, about 5 kb to about 20 kb, about 5 kb to about 15 kb, about 5 kb to about 10 kb, about 10 kb to about 100 kb, about 10 kb to about 50 kb, or about 10 kb to about 25 kb.

[0273] In some embodiments, the present disclosure provides an HSV vector comprising a capsid protein and a nucleic acid comprising an identifier. In some embodiments, the present disclosure provides an HSV vector comprising a capsid protein and a nucleic acid comprising an identifier and an HSV viral repeat sequence (e.g., TR L / IR L and / or IR S / TR SIn some embodiments, the present disclosure provides an HSV vector comprising a capsid, an envelope, and a nucleic acid comprising an identifier. In some embodiments, the present disclosure provides an HSV vector comprising a capsid, an envelope, and a nucleic acid comprising an identifier and an HSV viral repeat sequence (e.g., a TR L / IR L and / or IR S / TR S In some embodiments, the HSV vector comprises two viral repeat sequences including a terminal alpha sequence.

[0274] In some embodiments, the HSV nucleic acid comprises a payload, an identifier, and one or more sequences obtained or derived from an HSV virus. In some embodiments, the HSV vectors produced by the methods and / or insect cells of the disclosure comprise an HSV capsid, an envelope, and a nucleic acid comprising a payload, an identifier, and one or more sequences obtained or derived from an HSV virus (e.g., TR L / IR L and / or IR S / TR S In some embodiments, the HSV vector comprises an HSV capsid, an envelope, and an HSV viral repeat sequence (e.g., TR L / IR L and / or IR S / TR S The nucleic acid sequence comprises a nucleic acid sequence flanked by a payload and / or identifier.

[0275] In some embodiments, insect cells in the insect cell library are genetically modified to contain one or more nucleic acid sequences essential for the production of an HSV vector. In some embodiments, insect cells in the insect cell library can have one or more HSV vector components provided. In some embodiments, one or more components of the HSV vector are under the control of an inducible transcriptional control element (e.g., a promoter and / or enhancer). In some embodiments, insect cells (e.g., in a population of insect cells) contain (i) an identifier disposed between two viral repeat sequences, and (ii) one or more polynucleotides essential for the production of an HSV vector, and the insect cell(s) produce an HSV vector containing the identifier. In some embodiments, the HSV vector comprises a payload. In some embodiments, the identifier and / or payload, or a portion thereof, are subsequently removed from the HSV vector. In certain embodiments, where the identifier and / or payload, or a portion thereof, has been removed from the HSV vector, the payload, or a portion thereof, is replaced with a different payload, or a portion thereof.

[0276] Baculovirus vectors Baculoviruses are the most common viruses known to affect insect populations. They are double-stranded, circular, supercoiled DNA molecules contained within a rod-shaped capsid. Baculoviruses have double-stranded, circular DNA genomes generally between 80 kb and 200 kb in length.

[0277] Classically, baculoviruses are divided into two morphologically distinct genera: nuclear polyhedrosis viruses (NPVs) and granulosis viruses. Modern classification generally divides baculoviruses into four genera: αbaculoviruses (Lepidoptera-specific NPVs), βbaculoviruses (Lepidoptera-specific granulosis viruses), γbaculoviruses (Hymenoptera-specific NPVs), and Δbaculoviruses (Diptera-specific NPVs). In some embodiments, the baculovirus is Autographa californica multiple nucleopolyhedrovirus (AcMNPV) or a derivative thereof.

[0278] In some embodiments, the baculovirus expression vector comprises a payload, which in some embodiments is inserted into a non-essential genomic locus (e.g., the egt locus and / or the polyhedrin locus) of the baculovirus genome.

[0279] In some embodiments, a baculoviral vector is used to transfer one or more components of a viral vector into insect cells. For example, in some embodiments, a baculoviral vector can include one or more sequences from an AAV vector in its genome. In some embodiments, a baculoviral vector includes an AAV ITR sequence and an identifier. In some embodiments, a baculoviral vector including an AAV ITR sequence and an identifier (e.g., located between the ITR sequences) can further include a payload and / or one or more library variants.

[0280] Library construction The present disclosure provides library constructs for modifying insect cells according to the present disclosure. In some embodiments, the library constructs can contain two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more modified sequences. In some embodiments, the library constructs can contain up to 100 modified sequences.

[0281] The library construct comprises at least an identifier and a gene architecture suitable for packaging the identifier into a viral vector. As used herein, the term library construct also includes any library variant. In some embodiments, the library construct further comprises a payload for packaging into the viral vector (e.g., between viral repeat sequences). For example, in some embodiments, the library construct comprises (i) an identifier and a payload disposed between viral repeat sequences, and (ii) at least one modified sequence comprising at least one library variant.

[0282] In some embodiments, the library construct further comprises one or more constructs comprising cis-acting integration sequences (e.g., homology arms, recognition sites, and / or viral repeat sequences). For example, in some embodiments, the library construct comprises (i) an identifier and a payload disposed between the viral repeat sequences, (ii) at least one modified sequence comprising at least one library variant, and (iii) a cis-acting integration sequence for integrating the library construct, or a portion thereof, into the insect cell genome.

[0283] In some embodiments, the library construct further comprises at least one barcode. For example, in some embodiments, the library construct comprises (i) an identifier and a payload disposed between the viral repeat sequences, (ii) at least one modified sequence comprising at least one library variant, and (iii) at least one barcode. As another example, in some embodiments, the library construct comprises (i) an identifier and a payload disposed between the viral repeat sequences, (ii) at least one modified sequence comprising at least one library variant, (iii) at least one barcode, and (iv) a cis-acting integration sequence for integrating the library construct, or a portion thereof, into the insect genome.

[0284] In some embodiments, the library construct further comprises at least one modified sequence comprising at least one reporter and / or selectable marker. In some embodiments, one or more polynucleotides comprising the library construct comprise a reporter and / or selectable marker. In the context of the present disclosure, any suitable reporter (e.g., GFP, RFP, YFP, lacZ, etc.) or selectable marker (e.g., one that confers an artificially selectable trait, such as a resistance cassette) can be used.

[0285] Continuity of library construction The library construct may be a single contiguous construct or multiple discontinuous constructs. In some embodiments, the library construct is a single (i.e., one) contiguous construct. In such embodiments having a single contiguous library construct, characterization of the resulting viral vector directly provides information about any library variant (e.g., determining the identifier of the viral vector can be directly associated with any library variant). In some embodiments, the library construct comprises multiple discontinuous constructs. In such embodiments in which the library construct is a discontinuous library construct, the provided methods also include identifying the library variants in insect cells (e.g., by single-cell sequencing).

[0286] In some embodiments, the insect cell population comprises a plurality of library constructs, each individual library construct consisting of a single contiguous nucleic acid sequence, and the plurality of library constructs comprises a plurality of unique nucleic acid sequences. In some embodiments, the insect cell population comprises a plurality of library constructs, each individual library construct consisting of a discontinuous nucleic acid sequence, and the plurality of library constructs comprises a plurality of unique nucleic acid sequences.

[0287] In some embodiments, the insect cells provided comprise a library construct comprising a plurality of polynucleotides, wherein each individual insect cell comprises exactly one unique polynucleotide from a first subset of the plurality of polynucleotides comprising the library construct and two or more unique polynucleotides from a second subset of the plurality of polynucleotides comprising the library construct. For example, in some embodiments, each individual insect cell comprises exactly one unique identifier and two or more unique library variants. In some embodiments, the insect cells provided comprise a library construct comprising a plurality of polynucleotides, wherein each individual insect cell comprises exactly two unique polynucleotides from the first subset of the plurality of polynucleotides comprising the library construct and a plurality of unique polynucleotides from the second subset of the plurality of polynucleotides comprising the library construct. For example, in some embodiments, each individual insect cell comprises exactly two unique identifiers and a plurality of unique library variants.

[0288] Sequential library construction In some embodiments, the library construct is a single contiguous construct comprising at least one identifier flanked by genetic architecture suitable for packaging the identifier into a viral vector (e.g., viral repeat sequences, e.g., AAV ITR sequences), and any library variants. In some embodiments, the single contiguous library construct comprises an identifier and one or more library variants, where both the identifier and the library variant are positioned between the viral repeat sequences. In some embodiments, the single contiguous library construct comprises an identifier positioned between the viral repeat sequences and one or more library variants positioned outside the viral repeat sequences. In some embodiments, the single contiguous library construct comprises an identifier and one or more library variants positioned between the viral repeat sequences, and one or more additional library variants positioned outside the viral repeat sequences. In some embodiments, the library construct is a contiguous library construct and comprises a reporter and / or selectable marker. Some exemplary configurations of single contiguous library constructs are shown in Table 1 below.

[0289] [Table 1-1] [Table 1-2]

[0290] Subcellular location and copy number In some embodiments, the library construct is episomal and / or integrated into the insect cell genome. In some embodiments, the library construct is a single contiguous construct that is episomal. In some embodiments, the library construct is a single contiguous construct that is integrated into the insect cell genome. In some embodiments, the library construct further comprises cis-acting integration sequences for integration into the insect cell genome. For example, any of the exemplary embodiments in Table 1 may further comprise cis-acting integration sequences (e.g., homology arms, recognition sites, and / or viral repeat sequences).

[0291] In some embodiments, a single contiguous library construct is integrated into the insect cell genome. In some embodiments, the single contiguous library construct comprises a cis-acting integration sequence. In some embodiments, the single contiguous library construct comprises cis-acting integration sequences located at the 3' and 5' ends of the library construct. In some embodiments, the single contiguous library construct is integrated into the insect cell genome at low copy number (e.g., 10 or fewer copies of the library construct). In some embodiments, 4 or fewer copies of the single contiguous library construct are integrated into the insect cell genome. In some embodiments, 3 or fewer copies of the single contiguous library construct are integrated into the insect cell genome. In some embodiments, 2 or fewer copies of the single contiguous library construct are integrated into the insect cell genome. In some embodiments, a single copy of the single contiguous library construct is integrated into the insect cell genome.

[0292] In some embodiments, the single contiguous library construct is present episomally in the insect cell. In some embodiments, the single contiguous library construct is present episomally in the insect cell at low copy number (e.g., 10 copies or less of the library construct, e.g., 4 copies or less of the library construct, e.g., 3 copies or less of the library construct, 2 copies or less of the library construct, e.g., a single (1) copy of the library construct).

[0293] Discontinuous library construction In some embodiments, the provided methods and cells include discontinuous library constructs, e.g., allowing for simultaneous screening of multiple library variants. In some embodiments, the library constructs include multiple discontinuous constructs, at least one of which includes an identifier and a gene architecture suitable for packaging the identifier into a viral vector. In some embodiments, the discontinuous library constructs include a first construct including an identifier disposed between viral repeat sequences and one or more additional constructs. In some embodiments, the discontinuous library constructs include a first construct including an identifier and a payload disposed between viral repeat sequences and one or more additional constructs including one or more library variants. In some embodiments, the discontinuous library constructs include a first construct including, e.g., any of the single contiguous library constructs listed in Table 1, and one or more additional constructs (e.g., including additional library variants).

[0294] In some embodiments, the additional discontinuous library constructs comprise components of a viral vector, such as a viral Cap gene and a barcode. In some embodiments, the library comprises library constructs with viral vector components (e.g., Cap genes) of modified or different serotypes. For example, a library of viral Cap genes may be simultaneously screened with Cap genes of different serotypes (e.g., to select insect cells with improved characteristics of viral vectors of multiple different serotypes).

[0295] In certain embodiments, the one or more additional constructs comprise a library variant (e.g., of a modified or different serotype) that is a variant viral vector component. In some embodiments, the library construct comprises a library construct described in Table 1 and one or more additional constructs, wherein at least one additional construct comprises a viral Cap gene and a barcode.

[0296] In some embodiments, multiple separate libraries of library variants are screened, each associated with a separate identifier. For example, a library of gRNA library variants and a library of Cap gene variants can be screened simultaneously, allowing for the selection of gRNA variants that enrich for different Cap gene library variants (e.g., to select for gRNA-mediated perturbations that have beneficial effects in different viral vector serotypes).

[0297] In some embodiments, the library construct further comprises one, two, three, four, five, or more constructs, each comprising one or more library variants. In some embodiments, one or more additional constructs comprising one or more library variants and / or the construct comprising the identifier further comprise one or more barcodes.

[0298] In some embodiments, the library construct is a discontinuous library construct (e.g., individual construct) that comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 discontinuous nucleic acid sequences. In some embodiments, the library construct is a discontinuous library construct that comprises up to 20 discontinuous nucleic acid sequences, up to 30 discontinuous nucleic acid sequences, up to 40 discontinuous nucleic acid sequences, up to 50 discontinuous nucleic acid sequences, up to 60 discontinuous nucleic acid sequences, up to 70 discontinuous nucleic acid sequences, up to 80 discontinuous nucleic acid sequences, up to 90 discontinuous nucleic acid sequences, or up to 100 discontinuous nucleic acid sequences.

[0299] In some embodiments, the library construct is discontinuous and one or more individual polynucleotides of the library construct comprise a reporter and / or a selectable marker. In some embodiments, the library construct is discontinuous and a plurality of individual polynucleotides of the library construct comprise a reporter and / or a selectable marker. In some embodiments, the library construct is discontinuous and each of the individual polynucleotides of the library construct comprises a reporter and / or a selectable marker.

[0300] [Table 2]

[0301] Subcellular location and copy number In some embodiments, the library constructs are discontinuous and one or more individual constructs are episomal. In some embodiments, the library constructs are discontinuous and one or more individual constructs are integrated into the insect cell genome. In some embodiments, the library constructs are discontinuous and at least one construct is episomal and at least one construct is integrated into the insect cell genome.

[0302] In some embodiments, one or more individual constructs of the discontinuous library construct are integrated into the insect cell genome. In some embodiments, one or more individual constructs of the discontinuous library construct comprise a cis-acting integration sequence. In some embodiments, one or more individual constructs of the discontinuous library construct comprise a cis-acting integration sequence located at the 3' and 5' ends of each construct. In some embodiments, the cis-acting integration sequence comprises a viral repeat sequence (e.g., positioned outside any viral repeat sequences for packaging into a viral vector).

[0303] In some embodiments, where the library construct comprises multiple, non-contiguous constructs, one or more of the individual constructs are present episomally in the insect cell. In some embodiments, one or more of the individual constructs are present episomally in the insect cell at low copy number (e.g., 10 or fewer copies of the library construct, e.g., 4 or fewer copies of the library construct, e.g., 3 or fewer copies of the library construct, 2 or fewer copies of the library construct, e.g., a single (1) copy library construct).

[0304] identifier The present disclosure provides identifiers that can detect and identify insect cells or clonal cell lines from which viral vectors are produced and / or derived. In some embodiments, the relative abundance of a particular identifier can be characterized, detected, and / or quantified among pooled viral vector samples (e.g., each containing the identifier). In some embodiments, at least some of the identifiers are detected. In some embodiments, the identifiers are detected by sequencing (e.g., by next-generation sequencing). For example, in some embodiments, at least some of the identifiers are detected by next-generation sequencing and / or single-cell sequencing and / or Sanger sequencing.

[0305] In some embodiments, the identifier is present in the insect cell and also in the viral vector expressed by the insect cell. In some embodiments, the present disclosure provides an identifier present in a library construct. In some embodiments, the identifier is located between sequences for packaging into the viral vector (e.g., viral repeat sequences, e.g., AAV ITRs for expression of AAV vectors). In some embodiments, the viral vectors expressed by the insect cells described herein comprise an identifier, for example, in the nucleic acid of the viral vector. In some embodiments, the viral vectors expressed by the insect cells described herein comprise a nucleic acid comprising an identifier located between two viral repeat sequences (e.g., ITRs of an AAV vector). In some embodiments, at least a portion of the identifier in the nucleic acid of the viral vector is detected by next-generation sequencing and / or single-cell sequencing and / or Sanger sequencing.

[0306] In some embodiments, the insect cell expressing the viral vector and the expressed viral vector both contain the same identifier. In some embodiments, the identifier of the viral vector corresponds to the identifier of the insect cell that produced it. In some embodiments, the identifier of the viral vector is derived from the identifier of the insect cell that produced it. In some embodiments, the identifier of the viral vector and the identifier of the insect cell that produced the viral vector are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical.

[0307] In some embodiments, the identifier comprises a barcode. In some embodiments, the identifier is or comprises a barcode and is present in a library construct located between sequences for packaging into a viral vector (e.g., viral repeat sequences, e.g., AAV ITRs for expression of AAV vectors). In some embodiments, the identifier is or comprises a barcode present in a viral vector nucleic acid. In some embodiments, the provided methods comprise detecting an identifier comprising a barcode from a sample of viral vectors.

[0308] In some embodiments, the identifier comprises a library variant. In some embodiments, the identifier is present in a library construct that comprises the library variant and is disposed between sequences for packaging into a viral vector (e.g., viral repeat sequences, e.g., AAV ITRs for expression of an AAV vector). In some embodiments, the identifier comprises a library variant present in nucleic acid of the viral vector. In some embodiments, the provided methods comprise detecting an identifier comprising the library variant from a sample of the viral vector.

[0309] In some embodiments, the identifier comprises a barcode and a library variant. In some embodiments, the identifier is present in a library construct that comprises the barcode and the library variant and is disposed between sequences for packaging into a viral vector (e.g., viral repeat sequences, e.g., AAV ITRs for expression of an AAV vector). In some embodiments, the identifier comprises a barcode and a library variant that is present in nucleic acid of the viral vector. In some embodiments, provided methods comprise detecting at least a portion of the identifier that comprises the barcode and the library variant from a sample of the viral vector.

[0310] Barcode The present disclosure encompasses the recognition that barcodes can be useful for tracking associations between different components. In some embodiments, the barcode is a type of modified sequence. In some embodiments, the barcode is a type of modified nucleic acid sequence. In some embodiments, the barcode is part of a library construct.

[0311] In some embodiments, the library construct comprises one or more barcodes that, upon detection (e.g., by next-generation sequencing), indicate the identity of one or more library variants and / or other modified sequences not directly detected. In some embodiments, one barcode is associated with one or more modified sequences. For example, in some embodiments, one barcode is associated with one or more library variants. In some embodiments, one barcode is associated with one modified sequence. For example, in some embodiments, one barcode is associated with one library variant.

[0312] In some embodiments, the barcode does not comprise an identifier. In some embodiments, the barcode comprises an identifier. In some embodiments, the barcode comprises an identifier comprising a nucleic acid sequence. In some embodiments, the barcode comprises an identifier comprising a modified sequence. In some embodiments, the identifier does not comprise a barcode. In some embodiments, the identifier comprises a barcode. In some embodiments, the identifier comprises a barcode comprising a nucleic acid sequence. In some embodiments, the identifier comprises a barcode comprising a modified sequence. In some embodiments, the insect cell comprises multiple barcodes, at least one barcode is an identifier and at least one barcode is not an identifier.

[0313] In some embodiments, the library construct comprises a barcode. In some embodiments, the barcode is used as an identifier. In some embodiments, the barcode is used as an identifier and is placed between two viral repeat sequences. In some embodiments, the barcode is not used as an identifier. In some embodiments, the barcode is not used as an identifier and is placed between two viral repeat sequences but is not detected (e.g., by next-generation sequencing). In some embodiments, the library construct comprises a barcode sequence placed outside the two viral repeat sequences and therefore is not packaged into a viral vector.

[0314] In some embodiments, barcodes are used as identifiers, where the barcodes are present in a library construct located between sequences for packaging into a viral vector (e.g., viral repeat sequences, e.g., AAV ITRs for expression of AAV vectors). In some embodiments, barcodes are used as identifiers that allow identification of the insect cells or clonal cell lines in which viral vectors are produced and / or derived. In some embodiments, the relative abundance of a barcode indicates the relative productivity of the insect cells from which it is derived. In some embodiments, barcodes are used as identifiers that, upon detection (e.g., by next-generation sequencing), also indicate the identity of one or more library variants and / or modified sequences that are not directly detected.

[0315] In some embodiments, the barcode is not used as an identifier, but rather indicates, upon detection (e.g., by next-generation sequencing and / or single-cell sequencing), the identity of one or more library variants and / or modified sequences that are not directly detected. For example, in some embodiments where the library construct is discontinuous, the barcode may be used to track one or more library variants. In some embodiments, the discontinuous library construct includes a first construct including an identifier disposed between viral repeat sequences and one or more additional constructs, each including a barcode. In some embodiments, the library construct further includes one, two, three, four, five, or more constructs, each including one or more library variants, and each individual construct further includes a barcode. In some embodiments, each library variant is associated with a unique barcode. As another example, in some embodiments where the library construct is contiguous, the barcode may also be used to track one or more library variants. In some embodiments, the contiguous library construct includes an identifier disposed between viral repeat sequences. In some embodiments, the library construct further includes one or more library variants and one or more barcodes. In some embodiments, each library variant is associated with a unique barcode.

[0316] In some embodiments, the barcode comprises a nucleic acid sequence having a length in the range of 3 nucleotides to 50 nucleotides. In some embodiments, the barcode comprises a nucleic acid sequence having a length within a range bounded by a lower limit and an upper limit, where the upper limit is greater than the lower limit. In some embodiments, the lower limit may be about 5 nucleotides, about 6 nucleotides, about 7 nucleotides, about 8 nucleotides, about 9 nucleotides, about 10 nucleotides, about 11 nucleotides, about 12 nucleotides, about 13 nucleotides, about 14 nucleotides, or about 15 nucleotides. In some embodiments, the upper limit may be about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 45 nucleotides, or about 50 nucleotides. In some specific embodiments, the barcode comprises a sequence having a length in the range of 5 nucleotides to 25 nucleotides. In some specific embodiments, the barcode comprises about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, or about 25 nucleotides. In some embodiments, the barcode comprises DNA and / or RNA. In some embodiments, the barcode comprises a DNA sequence having a length in the range of 3 nucleotides to 50 nucleotides, or about 5 nucleotides to about 25 nucleotides. In some embodiments, the barcode comprises an RNA sequence having a length in the range of 3 nucleotides to 50 nucleotides, or about 5 nucleotides to about 25 nucleotides.

[0317] In some embodiments, the provided methods include detecting the barcode (e.g., by sequencing, e.g., by next generation sequencing and / or single cell sequencing and / or Sanger sequencing).

[0318] Library Variants The present disclosure provides insect cells comprising one or more library variants that create perturbations that vary among insect cells of the library. In some embodiments, the library variants create perturbations that can affect specific characteristics of viral vector production. In some embodiments, the library variants include modified sequences that create perturbations. In some embodiments, the library variants are sequence changes. In some embodiments, the library variants are epigenetic changes. In some embodiments, the library variants are effectors, whereby the library variants affect or create perturbations that vary among cells. In some embodiments, the library variants themselves can be perturbations that vary among cells. For example, in some embodiments, a library variant that is a gRNA is an effector that, together with an RNA-guided nuclease (e.g., a perturbation accessory sequence), creates a deletion in the genomic DNA of a cell. In other embodiments, the library variants are ORFs or gene sequences that, upon transfection into a cell and, in some cases, upon integration into genomic DNA (e.g., via trans- and cis-acting integration sequences), themselves result in perturbations or modifications to the genetic material of a cell.

[0319] In some embodiments, the library variants comprise a guide RNA sequence. In some embodiments, the library variants comprising the guide RNA sequence can also be an identifier (e.g., a unique gRNA sequence that associates the viral vector with the insect cell that produced it). In some embodiments, the insect cell comprises a library variant comprising a guide sequence that can be incorporated by the viral vector. In some embodiments, the guide sequence is approximately 10-30 nucleotides in length. In some embodiments, the guide sequence is 15-25 nucleotides in length. In certain embodiments, the guide sequence is 16-24 nucleotides in length (e.g., 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides in length). In some embodiments, the guide sequence is at or proximal to the 5' end of the gRNA (e.g., with Cas9 or a nuclease derived or derived therefrom). In some embodiments, the guide sequence is at or proximal to the 3' end of the gRNA (e.g., with Cpf1 or a nuclease derived or derived therefrom).

[0320] In some embodiments, the library variants encode guide RNA sequences associated with the introduction of genomic deletions. In some embodiments, the library variants encode guide RNA sequences associated with the introduction of genomic variations (e.g., SNPs). In some embodiments, the library variants encode guide RNA sequences associated with the introduction of genomic rearrangements. In some embodiments, the library variants encode guide RNA sequences associated with altered expression (e.g., activation and / or repression) of genes.

[0321] In some embodiments, the library variants comprise one or more ORFs. In some embodiments, the library variants comprise an ORF. In some embodiments, the library variants comprise an ORF that encodes an RNA sequence. In some embodiments, the ORF encodes a polypeptide (e.g., a protein, e.g., a glycoprotein). In some embodiments, the ORF encodes a fusion polypeptide and / or a chimeric polypeptide.

[0322] In some embodiments, the library variants comprise one or more genes. In some embodiments, the library variants comprise genes. In some embodiments, the library variants comprise genes that encode RNA sequences. In some embodiments, the genes encode polypeptides (e.g., proteins, e.g., glycoproteins). In some embodiments, the genes encode fusion and / or chimeric polypeptides. In some embodiments, the library variants comprise insect genes. In some embodiments, the library variants comprise viral genes (e.g., Cap genes).

[0323] In some embodiments, the library variants encode non-coding nucleic acid sequences, hi some embodiments, the library variants encode regulatory RNA sequences (e.g., siRNAs, microRNAs, etc.).

[0324] In some embodiments, a library variant or portion thereof is also an identifier, although a library construct may include one or more additional library variants that are not identifiers. In some embodiments, a library variant or portion thereof is an identifier, and the library variant is placed between sequences for packaging into a viral vector (e.g., viral repeat sequences, e.g., AAV ITRs for expression of an AAV vector). In some embodiments, the library variant is not an identifier (e.g., is not packaged into a viral vector). In some embodiments, where a library variant or portion thereof is an identifier, the library variant or portion thereof is unique to that particular insect cell or cell line (and the viral vector expressed therefrom).

[0325] In some embodiments, the library variants include components of viral vectors, such as modified components of viral vectors and / or components of various serotypes. In this manner, viral vectors of various modifications or serotypes may be screened. In some embodiments, the library variants include Cap genes (e.g., of various serotypes).

[0326] In some embodiments, provided insect cells individually comprise at least one library variant, wherein at least one library variant comprises at least one modified sequence comprising at least one gene, at least one ORF, at least one gRNA sequence, at least one unique non-coding nucleic acid sequence, or a combination and / or a plurality thereof. In some embodiments, an insect cell or insect cell population comprises a plurality of library variants, wherein the plurality of library variants comprises at least one modified sequence comprising at least one unique gene, at least one unique ORF, at least one unique gRNA sequence, and / or at least one unique non-coding nucleic acid sequence, or a combination and / or a plurality thereof.

[0327] In some embodiments, provided insect cells comprise one or more library variants. In some embodiments, provided insect cells comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more library variants. In some embodiments, provided insect cells comprise at least 100 library variants. In some embodiments, provided insect cells comprise about 2 to about 100 library variants, about 2 to about 20 library variants, about 3 to about 30 library variants, about 4 to about 40 library variants, or about 5 to about 50 library variants. In some embodiments, provided insect cells comprise 10 or fewer library variants, 20 or fewer library variants, 30 or fewer library variants, 40 or fewer library variants, 50 or fewer library variants, 60 or fewer library variants, 70 or fewer library variants, 80 or fewer library variants, 90 or fewer library variants, or 100 or fewer library variants.

[0328] In some embodiments, the library construct comprises at least one library variant and at least one identifier, wherein both the at least one library variant and the at least one identifier are positioned between two viral repeat sequences. In some embodiments, the library construct comprises at least one library variant and at least one identifier, wherein the at least one identifier is positioned between two viral repeat sequences, and the at least one library variant is positioned outside the two viral repeat sequences. In some embodiments, the library construct comprises at least two library variants and one identifier, wherein the identifier and the at least one library variant are positioned between two viral repeat sequences, and the at least one library variant is positioned outside the two viral repeat sequences. In some embodiments, the library construct further comprises one or more additional modified sequences positioned between and / or outside the two viral repeat sequences. For example, in some embodiments, the library construct further comprises a payload positioned between two viral repeat sequences. As another example, in some embodiments, the library construct further comprises one or more additional barcodes positioned between and / or outside the two viral repeat sequences.

[0329] In some embodiments, the library construct comprises an identifier that is a library variant and one or more additional library variants. In some embodiments, the library construct is a single contiguous library construct comprising an identifier that is a library variant located between sequences for packaging into a viral vector (e.g., viral repeat sequences, e.g., AAV ITRs for expression of an AAV vector) and one or more additional library variants located outside the sequences for packaging into a viral vector. In some embodiments, the library construct is a discontinuous library construct comprising an identifier that is a library variant located between sequences for packaging into a viral vector (e.g., viral repeat sequences, e.g., AAV ITRs for expression of an AAV vector) and one or more additional constructs comprising one or more additional library variants. In some specific embodiments, the one or more additional library variants comprise a component of a viral vector (e.g., a Cap gene).

[0330] In some embodiments, the library construct comprises an identifier that is a barcode and one or more library variants. In some embodiments, the library construct is a single contiguous library construct comprising an identifier that is a barcode located between sequences for packaging into a viral vector (e.g., viral repeat sequences, e.g., AAV ITRs for expression of an AAV vector) and one or more library variants located outside the sequences for packaging into a viral vector. In some embodiments, the library construct is a discontinuous library construct comprising an identifier that is a barcode located between sequences for packaging into a viral vector (e.g., viral repeat sequences, e.g., AAV ITRs for expression of an AAV vector) and one or more additional constructs comprising one or more library variants.

[0331] Some exemplary configurations of library variants in library constructions are shown in Table 3 below.

[0332] [Table 3-1] [Table 3-2]

[0333] In some embodiments, the provided methods include detecting one or more library variants (e.g., by sequencing, e.g., by next generation sequencing).

[0334] Viral repeat sequences The present disclosure provides architecture suitable for packaging identifiers into viral vectors, such as viral repeat sequences. In some embodiments, the viral repeat sequence is a DNA and / or RNA sequence. In some embodiments, the viral repeat sequence is a DNA sequence. In some embodiments, the viral repeat sequence is an RNA sequence. In some embodiments, any nucleic acid sequence located between two viral repeat sequences is packaged into the viral vector.

[0335] In some embodiments, the viral repeat sequences are derived from the same type of virus as the target viral vector. For example, in some embodiments, the target viral vector is an AAV vector, and the viral repeat sequences are derived from AAV. In some embodiments, the viral repeat sequences are derived from the same viral strain as the target viral vector. For example, in some embodiments, the target viral vector is an AAV5 vector, and the viral repeat sequences are AAV5-derived ITRs. In some embodiments, the viral repeat sequences are derived from a different viral strain than the target viral vector, but can still be packaged into the target viral vector. In some embodiments, the viral repeat sequences are modified viral repeat sequences (e.g., comprising sequences from two or more viruses). In some embodiments, the viral repeat sequences are synthetic viral repeat sequences (e.g., designed based on a consensus viral repeat sequence).

[0336] In some embodiments, the target viral vector is an AAV vector, and the viral repeat sequence comprises the sequence of AAV ITR or its derivative.In some embodiments, the viral repeat sequence is derived from a different AAV strain from the AAV vector, but can still be incorporated by the target viral vector.In some embodiments, the viral repeat sequence is a modified AAV ITR sequence.In some embodiments, the viral repeat sequence is a synthetic AAV ITR sequence.

[0337] In some embodiments, the target viral vector is an adenovirus vector, and the viral repeat sequence comprises the sequence of adenovirus ITR or its derivative. In some embodiments, the viral repeat sequence is derived from a different adenovirus strain from the adenovirus vector, but can still be incorporated by the target viral vector. In some embodiments, the viral repeat sequence is a modified adenovirus ITR sequence. In some embodiments, the viral repeat sequence is a synthetic adenovirus ITR sequence.

[0338] In some embodiments, the target viral vector is a lentiviral vector, and the viral repeat sequence comprises a sequence of a lentiviral LTR or a derivative thereof. In some embodiments, the target viral vector is an HIV-1 vector, and the viral repeat sequence comprises a sequence of an HIV-1 LTR or a derivative thereof. In some embodiments, the viral repeat sequence is derived from a different lentivirus strain than the target lentiviral vector, but is still capable of being incorporated by the target lentiviral vector. In some embodiments, the viral repeat sequence is a modified adenoviral ITR sequence. In some embodiments, the viral repeat sequence is a synthetic adenoviral ITR sequence.

[0339] In some embodiments, the targeted viral vector is an HSV vector and the viral repeat sequence is an HSV TR L / IR Land / or HSV IR S / TR S and / or derivatives thereof. In some embodiments, the targeted viral vector is an HSV-1 vector and / or an HSV-2 vector, and the viral repeat sequence comprises an HSV TR L / IR L and / or HSV IR S / TR S and / or derivatives thereof. In some embodiments, the viral repeat sequences are derived from a different HSV strain than the target HSV vector, but are still capable of being incorporated by the target HSV vector. In some embodiments, the viral repeat sequences are derived from a modified HSV TR L / IR L and / or HSV IR S / TR S In some embodiments, the viral repeat sequence is a synthetic HSV TR L / IR L and / or HSV IR S / TR S It is an array.

[0340] payload Among other things, the present disclosure provides viral vectors comprising a payload. The payload is generally any sequence of interest that one desires to introduce into a cell, organ, organism, and / or biological system (e.g., including a cell). For example, in some embodiments, the viral vector may comprise a payload that can be used to edit cells (e.g., encoding a genome editing tool for use in gene therapy and / or cell therapy). In some embodiments, the payload is included in a library construct. In some embodiments, the payload is included in a library construct and positioned between sequences for packaging into the viral vector (e.g., viral repeat sequences, e.g., AAV ITRs for expression of AAV vectors).

[0341] In some embodiments, the payload sequence comprises one or more of a coding region, a gene regulatory element, and a transcription terminator. Non-limiting examples of gene regulatory elements include promoters, transcription activators, enhancers, and polyadenylation signals. In some embodiments, the payload sequence comprises a coding region, a gene regulatory element, and a transcription terminator, which are arranged relative to each other such that the coding region is between the gene regulatory element and the transcription terminator.

[0342] In some embodiments, the coding region encodes a gene product. In some embodiments, the gene product is RNA. In some embodiments, the coding region encodes a polypeptide (e.g., a protein, e.g., a glycoprotein). In some embodiments, the coding region encodes a fusion polypeptide and / or chimeric polypeptide. In some embodiments, the coding region encodes one gene product. In some embodiments, the coding region encodes multiple gene products (e.g., 2, 3, 4, 5, 6, 7 or more gene products). In some embodiments, the payload sequence comprises a regulatory nucleic acid (e.g., tRNA, rRNA, tmRNA, miRNA, siRNA, piRNA, shRNA, antisense RNA, double-stranded RNA, snRNA, snoRNA, and / or long non-coding RNA (IncRNA), etc.). In some embodiments, the coding region encodes a regulatory RNA (e.g., siRNA, microRNA, etc.).

[0343] In some embodiments, the payload of the viral vectors described herein may be a gene therapy payload and may encode any protein or portion thereof that is beneficial to a subject, for example, a subject with a disease or disorder. The protein may be an extracellular, intracellular, or membrane-bound protein. In some embodiments, the protein encoded by the payload sequence includes, but is not limited to, a mammalian protein (e.g., a human protein).

[0344] In some embodiments, the encoded protein is or comprises a glycoprotein. In some embodiments, the encoded protein is or comprises an antibody and / or an Fc fusion protein. In some embodiments, the encoded protein is or comprises an antigen.

[0345] The protein can be a therapeutic protein. In some embodiments, the subject to whom gene therapy is administered has a disease or disorder whereby the subject's endogenous version of the protein is defective, produced in limited amounts, or not produced at all. In some such embodiments, the payload encodes a non-defective version of the protein. In some embodiments, the subject to whom gene therapy is administered has a disease or disorder mediated by a target gene (e.g., by the expression level of the target gene and / or the activity level of the target polypeptide), and the payload encodes an inhibitor of the target gene or target polypeptide. Examples of therapeutic proteins include, but are not limited to, injectable or injectable therapeutic proteins, enzymes, enzyme cofactors, hormones, blood or blood clotting factors, cytokines and interferons, growth factors, adipokines, and the like.

[0346] In some embodiments, the payload may include a gene-editing component. In some embodiments, the payload may include a nucleic acid to be excised (e.g., when the viral vector delivers an RNA-guided nuclease to the target cell). In some embodiments, the payload including the gene-editing component may encode any suitable endonuclease known in the art. For example, the payload may encode or include one or more components of a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas system.

[0347] The payload sequence can be any length compatible with the relevant viral vector. In some embodiments, the payload sequence is adjacent to one or more sequences obtained or derived from a virus (for example, the ITR sequence of AAV). In some embodiments, the payload sequence is placed between sequences for packaging into a viral vector. In some embodiments, the payload sequence is placed between viral repeat sequences (for example, the ITR sequence of AAV).

[0348] In some embodiments, the payload comprises a promoter. In some cases, the payload may comprise an enhancer sequence.

[0349] In some embodiments, any of the payloads described herein may include an untranslated region (UTR), such as 5'UTR or 3'UTR. The UTR of a gene is transcribed but not translated. The 5'UTR begins at the transcription start site and continues up to, but not including, the start codon. The 3'UTR begins immediately after the stop codon and continues up to the transcription termination signal.

[0350] In some embodiments, the protein-encoding payloads provided herein may contain a polyadenylation (poly(A)) signal sequence. Most new eukaryotic mRNAs have a poly(A) tail at their 3' end, which is added in a complex process involving cleavage of the primary transcript and coupled polyadenylation driven by the poly(A) signal sequence (e.g., Proudfoot et al., Cell 108:501-512, 2002, incorporated herein by reference in its entirety). The poly(A) tail confers stability and transmissibility to the mRNA (Molecular Biology of the Cell, Third Edition by B. Alberts et al., Garland Publishing, 1994, incorporated herein by reference in its entirety). In some embodiments, the poly(A) signal sequence is located 3' to the coding sequence.

[0351] In some embodiments, the protein-encoding payload may contain an internal ribosome entry site (IRES). IRESs form complex secondary structures, allowing translation initiation to occur from any position with the mRNA immediately downstream of where the IRES is located (see, e.g., Pelletier and Sonenberg, Mal. Cell. Biol. 8(3):1103-1112, 1988). There are several IRES sequences known to those skilled in the art, including those derived from foot-and-mouth disease virus (FMDV), encephalomyocarditis virus (EMCV), human rhinovirus (HRV), cricket paralysis virus, human immunodeficiency virus (HIV), hepatitis A virus (HAV), hepatitis C virus (HCV), and poliovirus (PV).

[0352] In some embodiments, any of the constructs provided herein can include splice donor and / or splice acceptor sequences that function during RNA processing that occurs during transcription. In some embodiments, the splice sites are involved in trans-splicing.

[0353] In some embodiments, the payloads provided herein may optionally include a sequence encoding a reporter polypeptide and / or reporter protein ("reporter sequence") and / or a sequence encoding a selectable marker (e.g., conferring a trait that can be artificially selected, such as, for example, a resistance cassette). Non-limiting examples of reporter sequences include DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), red fluorescent protein, mCherry fluorescent protein, yellow fluorescent protein, chloramphenicol acetyltransferase (CAT), and luciferase. Additional examples of reporter sequences are known in the art. When combined with control elements that drive their expression, reporter sequences can provide a signal that is detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescent, or other spectroscopic assays, fluorescence-activated cell sorting (FACS) assays, immunological assays (e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry). Similarly, when associated with the regulatory elements that drive their expression, selectable marker sequences can confer a trait that can also be artificially selected for by conventional means.

[0354] promoter The term "promoter" refers to a DNA sequence that is recognized by an enzyme / protein that can promote and / or initiate transcription of an operably linked coding sequence (e.g., a gene). For example, a promoter generally refers to a nucleotide sequence to which, for example, RNA polymerase and / or any associated factors can bind and initiate transcription therefrom. Thus, in some embodiments, the payload comprises a coding sequence operably linked to one of the non-limiting example promoters described herein.

[0355] In some embodiments, the promoter is functional in insect cells. In some embodiments, the promoter is a baculovirus ie1 promoter. In some embodiments, the promoter is a promoter derived from the baculovirus polyhedrin (polh) gene, p6.9 gene, and / or p10 gene.

[0356] In some embodiments, the promoter is an inducible promoter, a constitutive promoter, an insect cell promoter, a viral promoter, a chimeric promoter, a modified promoter, a tissue-specific promoter, or any other type of promoter known in the art.

[0357] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a coding sequence (e.g., a protein-coding sequence), causes transcription of RNA from a nucleic acid in a cell under most or all physiological conditions.

[0358] Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter (see, e.g., Boshart et al., Cell 41:521-530, 1985, which is incorporated herein by reference in its entirety), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFl-α promoter (Invitrogen).

[0359] Inducible promoters allow for the regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of specific physiological conditions, such as acute phase, a particular differentiation state of cells, or cell restriction during replication. Inducible promoters and induction systems are available from a variety of commercial sources, including, but not limited to, Invitrogen, Clontech, and Ariad. Additional examples of inducible promoters are known in the art.

[0360] Examples of inducible promoters regulated by exogenously supplied compounds include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system, the ecdysone insect promoter, the tetracycline-repressible system, the tetracycline-inducible system, the RU486-inducible system, and the rapamycin-inducible system.

[0361] The term "tissue-specific" promoter refers to a promoter that is active only in particular cell types and / or tissues (e.g., transcription of a particular gene occurs only in cells that express a transcriptional regulatory and / or control protein that binds to the tissue-specific promoter).

[0362] In some embodiments, the regulatory and / or control sequences confer tissue-specific gene expression capabilities. In some cases, the tissue-specific regulatory and / or control sequences bind tissue-specific transcription factors that induce transcription in a tissue-specific manner.

[0363] Enhancer The term "enhancer" refers to a nucleotide sequence that can increase the transcription level of a nucleic acid coding sequence (e.g., a protein). Enhancer sequences (generally 50-1500 bp in length) generally increase the level of transcription by providing additional binding sites for transcription-related proteins (e.g., transcription factors). In some embodiments, enhancer sequences are present within intron sequences. Unlike promoter sequences, enhancer sequences can act at locations much farther away from the transcription start site (e.g., compared to promoters). Non-limiting examples of enhancers include the RSV enhancer, CMV enhancer, CMV early enhancer, cAMP response element (CRE) enhancer, and / or SV40 enhancer.

[0364] In some embodiments, the enhancer comprises a baculovirus enhancer sequence (hr1).

[0365] Location of library construction elements relative to viral repeat sequences The library construct may comprise one or more modified sequences. In some embodiments, the library construct comprises a plurality of modified sequences. In some embodiments, the library construct comprises a viral repeat sequence for packaging a viral vector.

[0366] The one or more and / or plurality of modified sequences on the library construct can be at any number of positions and / or combinations of positions relative to each other and relative to the viral repeat sequences on the library construct. For example, in some embodiments, an insect cell or insect cell population comprises at least one library construct comprising a plurality of modified sequences, wherein a first subset of the plurality of modified sequences is located between the viral repeat sequences and a second subset of the plurality of modified sequences is located outside the viral repeat sequences.

[0367] In some embodiments, the plurality of modified sequences on the library construct comprises at least one library variant and at least one identifier. In some embodiments, all of the library variants and identifiers are positioned between viral repeat sequences. In some embodiments, the identifiers are positioned between viral repeat sequences, and all of the library variants are positioned outside of the viral repeat sequences. In some embodiments, the plurality of modified sequences comprises at least two library variants and at least one identifier. In some embodiments, the identifiers and at least one library variant are positioned between viral repeat sequences, and at least one library variant is positioned outside of the viral repeat sequences.

[0368] In some embodiments, the plurality of modified sequences comprises at least one library variant, at least one identifier, and at least one payload. In some embodiments, all library variants, identifiers, and payloads are positioned between viral repeat sequences. In some embodiments, all identifiers and payloads are positioned between viral repeat sequences, with all library variants positioned outside the viral repeat sequences. In some embodiments, the plurality of modified sequences comprises at least two library variants, at least one payload, and at least one identifier. In some embodiments, all identifiers, payloads, and at least one library variant are positioned between viral repeat sequences, with at least one library variant positioned outside the viral repeat sequences.

[0369] In some embodiments, the library construct further comprises at least one modified sequence comprising at least one reporter and / or selectable marker. In some embodiments, all reporters and / or selectable markers are positioned between viral repeat sequences. In some embodiments, all reporters and / or selectable markers are positioned outside viral repeat sequences. In some embodiments, the library construct comprises both at least one reporter and at least one selectable marker. In some embodiments, both at least one reporter and at least one selectable marker are positioned between viral repeat sequences. In some embodiments, both at least one reporter and at least one selectable marker are positioned outside viral repeat sequences. In some embodiments, at least one reporter is positioned between viral repeat sequences and at least one selectable marker is positioned outside viral repeat sequences. In some embodiments, at least one selectable marker is positioned between viral repeat sequences and at least one reporter is positioned outside viral repeat sequences.

[0370] In some embodiments, the library construct further comprises at least one modified sequence comprising at least one barcode. In some embodiments, all barcodes are positioned between viral repeat sequences. In some embodiments, all barcodes are positioned outside viral repeat sequences. In some embodiments, the library construct comprises multiple barcodes, a first subset of which is positioned between viral repeat sequences and a second subset of which is positioned outside viral repeat sequences.

[0371] In some embodiments, the library construct comprises at least one identifier and at least one barcode. In some embodiments, at least one barcode is used as an identifier, and in some embodiments, at least one barcode is not used as an identifier. In some embodiments, at least one barcode is placed between viral repeat sequences, and at least one barcode, or a portion thereof, is used as an identifier. In some embodiments, at least one barcode is placed between viral repeat sequences, and at least one barcode is not used as an identifier. In some embodiments, the library construct comprises at least one identifier and at least two barcodes. In some embodiments, all of the identifiers and at least one barcode are placed between viral repeat sequences, at least one barcode is placed outside the viral repeat sequences, and at least one barcode placed between the viral repeat sequences (or a portion thereof) is used as an identifier. In some embodiments, all of the identifiers and at least one barcode are placed between viral repeat sequences, at least one barcode is placed outside the viral repeat sequences, and none of the at least two barcodes are used as an identifier.

[0372] In some embodiments, the library construct comprises at least two barcodes, further comprises at least one additional modified sequence, and the barcode is not used as an identifier, but all barcodes are used to track the additional modified sequence. For example, in some embodiments, the library construct comprises at least two barcodes, further comprises at least one library variant, and the barcode is not used as an identifier, but all barcodes are used to track the library variant. In some embodiments, one barcode is used as an identifier, and the additional barcode is used to track the library variant.

[0373] In some embodiments, one barcode represents a single modified sequence (e.g., a single library variant). In some embodiments, one barcode represents multiple modified sequences (e.g., multiple library variants). In some embodiments, multiple barcodes represent a single modified sequence (e.g., a library variant). In some embodiments, multiple barcodes represent multiple modified sequences (e.g., multiple library variants). In some embodiments, no barcode is used as an identifier, and all barcodes are used to track other modified sequences that contain the barcode. In some embodiments, one barcode is used as an identifier, and additional barcodes are used to track other modified sequences that contain the barcode.

[0374] Use of constructs in insect cells Other features of the constructs and modified sequences promoter In some embodiments, the construct comprises a promoter. The term "promoter" refers to a DNA sequence recognized by an enzyme / protein capable of promoting and / or initiating transcription of an operably linked coding sequence (e.g., a gene). For example, a promoter generally refers to a nucleotide sequence to which, for example, RNA polymerase and / or any associated factors can bind and initiate transcription therefrom. Thus, in some embodiments, the modified sequence comprises a coding sequence operably linked to a promoter. A variety of promoters are known in the art, and such promoters may be used herein.

[0375] In some embodiments, the promoter is an inducible promoter, a constitutive promoter, an insect cell promoter, a viral promoter, a chimeric promoter, a modified promoter, a tissue-specific promoter, or any other type of promoter known in the art.

[0376] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a coding sequence (e.g., a protein-coding sequence), causes transcription of RNA from a nucleic acid in a cell under most or all physiological conditions.

[0377] Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter (see, e.g., Boshart et al., Cell 41:521-530, 1985, which is incorporated herein by reference in its entirety), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFl-α promoter (Invitrogen).

[0378] Inducible promoters allow for the regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of specific physiological conditions, such as acute phase, a particular differentiation state of cells, or cell restriction during replication. Inducible promoters and induction systems are available from a variety of commercial sources, including, but not limited to, Invitrogen, Clontech, and Ariad. Additional examples of inducible promoters are known in the art.

[0379] Examples of inducible promoters regulated by exogenously supplied compounds include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system, the ecdysone insect promoter, the tetracycline-repressible system, the tetracycline-inducible system, the RU486-inducible system, and the rapamycin-inducible system.

[0380] The term "tissue-specific" promoter refers to a promoter that is active only in particular cell types and / or tissues (e.g., transcription of a particular gene occurs only in cells that express a transcriptional regulatory and / or control protein that binds to the tissue-specific promoter).

[0381] In some embodiments, the regulatory and / or control sequences confer tissue-specific gene expression capabilities. In some cases, the tissue-specific regulatory and / or control sequences bind tissue-specific transcription factors that induce transcription in a tissue-specific manner.

[0382] Enhancer In some cases, the construct can include an enhancer sequence. The term "enhancer" refers to a nucleotide sequence that can increase the transcription level of a nucleic acid coding sequence (e.g., a protein). Enhancer sequences (generally 50-1500 bp in length) generally increase the level of transcription by providing additional binding sites for transcription-related proteins (e.g., transcription factors). In some embodiments, enhancer sequences are present within intron sequences. Unlike promoter sequences, enhancer sequences can act at locations much farther away from the transcription start site (e.g., compared to promoters). Non-limiting examples of enhancers include the RSV enhancer, CMV enhancer, CMV early enhancer, cAMP response element (CRE) enhancer, and / or SV40 enhancer.

[0383] Additional arrays In some embodiments, any of the constructs described herein may contain an untranslated region (UTR), such as a 5'UTR or a 3'UTR. The UTR of a gene is transcribed but not translated. The 5'UTR begins at the transcription initiation site and continues up to, but not including, the start codon. The 3'UTR begins immediately after the stop codon and continues to the transcription termination signal.

[0384] In some embodiments, constructs containing modified sequences encoding proteins provided herein may contain a polyadenylation (poly(A)) signal sequence. Most new eukaryotic mRNAs have a poly(A) tail at their 3' end, which is added in a complex process involving cleavage of the primary transcript and coupled polyadenylation driven by the poly(A) signal sequence (e.g., Proudfoot et al., Cell 108:501-512, 2002, incorporated herein by reference in its entirety). The poly(A) tail confers stability and transmissibility to the mRNA (Molecular Biology of the Cell, Third Edition by B. Alberts et al., Garland Publishing, 1994, incorporated herein by reference in its entirety). In some embodiments, the poly(A) signal sequence is located 3' to the coding sequence.

[0385] In some embodiments, constructs containing modified protein-encoding sequences may contain an internal ribosome entry site (IRES). IRESs form complex secondary structures, allowing translation initiation to occur from any position with the mRNA immediately downstream of where the IRES is located (see, e.g., Pelletier and Sonenberg, Mal. Cell. Biol. 8(3):1103-1112, 1988). There are several IRES sequences known to those skilled in the art, including those derived from foot-and-mouth disease virus (FMDV), encephalomyocarditis virus (EMCV), human rhinovirus (HRV), cricket paralysis virus, human immunodeficiency virus (HIV), hepatitis A virus (HAV), hepatitis C virus (HCV), and poliovirus (PV).

[0386] In some embodiments, any of the constructs provided herein can include splice donor and / or splice acceptor sequences that function during RNA processing that occurs during transcription. In some embodiments, the splice sites are involved in trans-splicing.

[0387] In some embodiments, any of the constructs provided herein may optionally include a sequence encoding a reporter polypeptide and / or reporter protein ("reporter sequence") and / or a sequence encoding a selectable marker (e.g., conferring a trait that can be artificially selected, such as, for example, a resistance cassette). Non-limiting examples of reporter sequences include DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), red fluorescent protein, mCherry fluorescent protein, yellow fluorescent protein, chloramphenicol acetyltransferase (CAT), and luciferase. Additional examples of reporter sequences are known in the art. When combined with control elements that drive their expression, reporter sequences can provide a signal that is detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescent, or other spectroscopic assays, fluorescence-activated cell sorting (FACS) assays, immunological assays (e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry). Similarly, when associated with the regulatory elements that drive their expression, selectable marker sequences can confer a trait that can also be artificially selected for by conventional means.

[0388] Insect cell modification and entry of cell constructs In some embodiments, the present disclosure provides a method comprising preparing or obtaining an insect cell library. Any method suitable for genetic modification can be used to manipulate insect cells for the present disclosure, for example, to introduce a library construct, to introduce another construct, and / or to introduce a perturbation. Numerous methods are known in the art for introducing exogenous nucleic acid (e.g., DNA) into eukaryotic cells, including, for example, transfection, infection (e.g., viral transduction), or electroporation.

[0389] Methods for delivering constructs into insect cells can vary depending on the needs. In certain embodiments, certain constructs may be delivered as nucleic acid (e.g., DNA) constructs in one or more plasmids. Delivery methods include, but are not limited to, electroporation, microinjection, gene guns, imparefection, hydrostatic pressure, continuous infusion, sonication, magnetofection, chemical vehicles (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-penetrating peptides), viral vectors (e.g., replication-competent viral vectors, replication-incompetent viral vectors, replication-defective viral vectors, replication-competent viral vectors, and / or replication-conditional viral vectors).

[0390] In some embodiments, the insect cell library is genetically modified to contain the library construct and / or other constructs. The library construct and / or other constructs may be delivered by any suitable method known to those skilled in the art of nucleic acid manipulation, including genetic modification, recombinant modification, and synthetic techniques (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, incorporated herein by reference in its entirety). In some embodiments, the provided methods include transfecting one or more nucleic acids comprising the library construct into the insect cells of the library.

[0391] In some embodiments, insect cell libraries are genetically modified to contain one or more nucleic acid sequences essential for viral vector production. In some embodiments, insect cell libraries that express and / or produce viral vectors may have one or more viral vector components provided to the insect cells in trans. For example, the recombinant AAV constructs and / or modified sequences, rep sequences, cap sequences, and helper functions required to produce the AAV vectors of the present disclosure may be delivered to packaging host cells using any suitable construct.

[0392] Constructs encoding viral elements may be delivered by any suitable method known to those skilled in the art of nucleic acid manipulation, including, for example, genetic modification, recombinant modification, and synthetic techniques (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, incorporated herein by reference in its entirety). In some embodiments, the methods provided comprise transfecting one or more nucleic acids comprising the viral construct or encoding the viral vector components into insect cells of the library. In some embodiments, the methods provided comprise transfecting one or more plasmid(s) comprising or consisting essentially of nucleic acid molecule(s) encoding the viral vector. In some embodiments, the viral vector components are comprised in one or more plasmids that are transfected into the cells. In some embodiments, the viral vector components are comprised in one, two, three, or more plasmids that are each transfected into the cells.

[0393] In some embodiments, baculovirus is used to infect insect cells with viral vector components, hi some embodiments, the viral vector components are contained on one or more baculovirus vectors that are transfected into insect cells.

[0394] In some embodiments, the viral vector is an adeno-associated virus (AAV), and viral transfection involves transfecting insect cells with a plasmid(s) containing or consisting essentially of a nucleic acid molecule(s) encoding the AAV vector, and providing the rep and / or cap of the AAV vector, which are essential for AAV vector replication and packaging. In some embodiments, one, two, three, or more plasmids each containing an AAV vector component are transfected into the cells. In some specific embodiments, the cells are transfected with plasmids (e.g., 1 to 5 plasmids, e.g., 3 plasmids) that, in combination, provide various AAV components, including a helper virus, a payload (e.g., a therapeutic gene), rep, and / or cap.

[0395] In certain embodiments, viral transfection of AAV constructs may be performed using a triple transfection method (e.g., as described in U.S. Pat. No. 6,001,650, incorporated herein by reference in its entirety). In some embodiments, AAV vectors are produced by transfecting host cells with one or more constructs containing one or more nucleic acid sequences essential for AAV vector production, including, but not limited to, rep and / or cap sequences, and / or constructs containing helper functions. In some embodiments, the rep and cap sequences function in trans for productive AAV vector replication and encapsidation. In some embodiments, constructs containing rep and / or cap sequences support efficient production of AAV constructs without producing detectable wild-type AAV vectors (i.e., AAV vectors containing functional rep and cap genes). The helper function construct encodes nucleotide sequences for non-AAV-derived viral and / or cellular functions, and the AAV vector is replication-dependent, which may include those functions required for replication of the AAV construct, including, but not limited to, portions involved in activation of AAV vector gene transcription, stage-specific AAV vector mRNA splicing, AAV DNA replication, synthesis of the cap expression product, and / or AAV vector capsid assembly. These viral-based functions can be obtained from any known helper virus, such as, for example, adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus.

[0396] In some embodiments, it may be beneficial to prevent the produced viral vector from reinfecting the insect cells of the insect cell library. In some embodiments, the insect cell(s) of the insect cell library have been modified to destroy or eliminate the receptor(s) of the produced viral vector. In some embodiments, the insect cells have been treated with an infection / blocking agent.

[0397] Location of constructs and modified sequences in insect cells Any of the constructs described herein may be present extrachromosomally in the insect cell and / or may be integrated into the insect cell genome.

[0398] In some embodiments, the library constructs are episomal and / or integrated into the insect cell genome. In some embodiments, the library constructs are a single contiguous construct that is episomal. In some embodiments, the library constructs are a single contiguous construct that is integrated into the insect cell genome. In some embodiments, the library constructs are discontinuous, and one or more individual constructs are episomal. In some embodiments, the library constructs are discontinuous, and one or more individual constructs are integrated into the insect cell genome. In some embodiments, the library constructs are discontinuous, and at least one construct is episomal, and at least one construct is integrated into the insect cell genome.

[0399] In some embodiments, one or more sequences essential for viral vector production are present extrachromosomally (e.g., episomally) in the insect cell, hi some embodiments, one or more sequences essential for viral vector production are integrated into the genome of the insect cell.

[0400] In some embodiments, one or more sequences essential for viral vector production are integrated into the genome of the insect cell, and one or more sequences are inducibly expressed. In some embodiments, all sequences essential for viral vector production are integrated into the genome of the insect cell, and one or more sequences are inducibly expressed. In some embodiments, all sequences essential for viral vector production are integrated into the genome of the insect cell, and two or more sequences are inducibly expressed. In some embodiments, all sequences essential for viral vector production are integrated into the genome of the insect cell, and all sequences are inducibly expressed.

[0401] Any method known in the art for integrating sequences can be used. In some embodiments, the integration is targeted integration (e.g., at a predetermined site). In some embodiments, the integration is random integration (e.g., at a random site). In some embodiments, the integration is random within a predetermined subset of genomic locations.

[0402] In some embodiments, one or more modified sequences (e.g., library constructs and / or other constructs) are pre-integrated into cells of an insect library using methods known in the art. In some embodiments, insect cells with one or more integrated sequences are transfected with a sequence encoding a viral vector. In some embodiments, one or more viral vector sequences are also integrated into the genome.

[0403] In some embodiments, insect cell libraries for culturing viral vectors may be stably modified to contain one or more such viral components (e.g., recombinant AAV constructs, rep sequences, cap sequences, and / or helper functions) using methods known to those of skill in the art. In some embodiments, such stable insect cells contain such viral vector component(s) under the control of an inducible promoter. In some embodiments, such viral vector component(s) may be under the control of a constitutive promoter. In some embodiments, selected stable insect cells may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable insect cell may contain E1 helper functions under the control of a constitutive promoter and rep and / or cap genes under the control of an inducible promoter. Other stable insect cells may be generated by those of skill in the art using routine methods as a basis for creating insect cell libraries. Such stably expressing insect cell lines may then be engineered to contain a variety of different modified sequences, including identifier sequences, thereby generating viral vector-producing insect cell libraries.

[0404] In some embodiments, the exogenous DNA (e.g., a library construct) is integrated into genomic DNA, thereby flanking the genomic DNA of the insect cell. In some embodiments, integration is mediated by natural DNA repair mechanisms endogenous to the cell. For example, integration can be achieved simply by introducing the exogenous DNA into the cell, allowing site-specific nucleases to create an integration site, and integrating the donor DNA. The cells may be maintained in culture for a time sufficient to allow the DNA to integrate. This typically results in a mixed population of cells containing (i) recombinant cells that have integrated the exogenous DNA at an integration site created by the site-specific nuclease, and optionally (ii) cells that have integrated the exogenous DNA at a site other than the desired integration site, and / or optionally (iii) cells that have not integrated the exogenous DNA. Selection methods known in the art may be used to enrich for cells harboring the genetic composition in the library.

[0405] Episomal library constructs In some embodiments, the present disclosure provides insect cells in which one or more constructs (e.g., library constructs and / or constructs encoding sequences essential for the production of viral vectors) are present episomally rather than integrated into the insect cell genome. Any method known in the art for introducing episomal constructs into insect cells can be used.

[0406] In some embodiments, one or more of the constructs is a baculovirus construct. In some embodiments, the library construct comprises a baculovirus construct.

[0407] In some embodiments, a single contiguous library construct is present episomally in insect cells. In some embodiments, one or more individual constructs of a discontinuous library construct are present episomally in insect cells (e.g., a construct comprising an identifier and a viral packaging sequence). In some embodiments, all individual constructs of a discontinuous library construct are present episomally in insect cells.

[0408] In some embodiments, exogenous nucleic acids (e.g., DNA) can be introduced into insect cells by standard transfection or electroporation methods. In some embodiments, exogenous nucleic acids introduced into insect cells can be present in the cells as episomes at single, low copy number, or high copy number. In some embodiments, exogenous nucleic acids introduced into insect cells can be present genomically and / or episomally at variable copy number. Cells can be enriched for episomal constructs (e.g., library constructs) using selection and / or screening methods known in the art.

[0409] In some embodiments, exogenous nucleic acid is introduced into insect cells using baculovirus expression system.In some embodiments, baculovirus construct promotes the transfer of nucleic acid into insect cells, and it is expressed as episome.In some specific embodiments, exogenous nucleic acid (e.g., DNA) encoding AAV genome (e.g., ITR sequence and adjacent sequence) is introduced into insect cells using baculovirus expression system.

[0410] In some embodiments, the pool of viral vectors produced by the first insect cell library is used to transfect insect cells to generate a second insect cell library. In some embodiments, the second insect cell library expresses the library variants as episomes. In some embodiments, the pool of viral vectors produced by the first insect cell library is a pool of AAV vectors. In some embodiments, the pool of AAV vectors produced by the first insect cell library is used to transduce insect cells to generate a second insect cell library containing the identifiers in episomes.

[0411] Integrated library constructs In some embodiments, the present disclosure provides insect cells having one or more constructs (e.g., constructs encoding sequences essential for the production of library constructs and / or viral vectors) integrated into the insect cell genome. Any method known in the art for integrating sequences can be used.

[0412] In some embodiments, integration of a construct (e.g., a library construct) into the insect cell genome is mediated by at least one trans-acting integration sequence. In some embodiments, the at least one trans-acting integration sequence comprises (i) an integration construct, (ii) a recombinase, (iii) a nuclease, (iv) a transposase, and / or a derivative thereof and / or a fusion thereof. In some embodiments, integration of a construct (e.g., a library construct) into the insect cell genome is mediated by at least one trans-acting integration sequence in coordination with at least one cis-acting integration sequence. In some embodiments, the cis-acting integration sequence comprises (i) viral repeat sequences, (ii) a recombinase recognition site, (iii) homology arms, and / or (iv) a transposase recognition site and / or a derivative thereof.

[0413] In some embodiments, the construct (e.g., library construct) to be integrated into the insect genome comprises at least one pair of cis-acting integration sequences flanking the sequence to be integrated. For example, a library construct is provided that comprises a first set of viral repeat sequences flanking the identifier and / or payload, the first set of viral repeat sequences flanking the cis-acting integration sequences.

[0414] In some embodiments, integration of the construct into the insect cell genome is targeted integration. In some embodiments, integration of the construct into the insect cell genome is random integration. In some embodiments, the construct is integrated into the insect cell genome at a random insertion site. In some embodiments, the random insertion site is random within a predetermined subset of genomic locations. In some embodiments, insertion of the library construct into the insect cell genome comprises a predetermined insertion site.

[0415] In some embodiments, a library construct (e.g., one or more individual nucleic acids associated with a library construct) is integrated into the insect genome. In some embodiments, a single contiguous library construct is integrated into the insect genome. In some embodiments, one or more individual constructs of a discontinuous library construct are integrated into the insect genome (e.g., a construct comprising an identifier and a viral packaging sequence). In some embodiments, all individual constructs of a discontinuous library construct are integrated into the insect genome.

[0416] In some embodiments, exogenous nucleic acids (e.g., DNA) can be introduced into insect cells by standard transfection or electroporation methods. In some embodiments, exogenous nucleic acids introduced into insect cells can be integrated into the genome at single, low, or high copy number and / or present episomally in the cells at single, low, or high copy number. In some embodiments, exogenous nucleic acids introduced into insect cells can be present genomically and / or episomally at variable copy number. Cells can be enriched for integrating constructs (e.g., library constructs) using selection and / or screening methods known in the art.

[0417] Viral transduction-mediated integration In some embodiments, the library construct is integrated into the insect genome via viral transduction-mediated integration. In some embodiments, a library construct is provided that includes, for example, a first set of viral repeat sequences flanking an identifier and / or payload, the first set of viral repeat sequences flanking a cis-acting integration sequence that includes a second set of viral repeat sequences. In some embodiments, the at least one trans-acting integration sequence comprises an integration construct, and the integration construct is an invertebrate parvovirus vector (e.g., Junonia coenia densovirus (JcDNV)) or a derivative thereof. See, e.g., Bossin H, Fournier P, Royer C, et al. Junonia coenia densovirus-based vectors for stable transgene expression in Sf9 cells: influence of the densovirus sequences on genomic integration. J Virol. 2003;77(20):11060-11071, which is incorporated herein by reference in its entirety.

[0418] Nuclease-mediated integration In some embodiments, the library construct is integrated into the insect genome by nuclease-mediated integration. In some embodiments, for example, a library construct is provided that includes a first set of viral repeat sequences flanking an identifier and / or a payload, the first set of viral repeat sequences flanking a cis-acting integration sequence that includes homology arms.

[0419] In some embodiments, at least one trans-acting integration sequence comprises a nuclease and / or a fusion thereof and / or a derivative thereof, including a modified Fok1 nuclease domain fusion to a programmable DNA-binding domain such as Cas9, CasZ, Cpfl, a TALE protein (TALEN) or a zinc finger protein (ZFN), and / or a meganuclease and / or a derivative thereof. In some embodiments, the nuclease is or comprises Cas9.

[0420] In some embodiments, at least one trans-acting integration sequence comprises a zinc finger nuclease. Zinc finger nucleases (ZFNs) are artificial restriction enzymes created by combining a zinc finger DNA binding domain with a DNA cleavage domain. The most common cleavage domain in ZFNs is the non-specific cleavage domain derived from the type IIs restriction endonuclease FokI, which can be targeted using a guide sequence. Methods for using ZFNs are described, for example, in WO2009146179A1, WO2008060510A2, and CN102174576A, which are incorporated by reference in their entirety.

[0421] In some embodiments, at least one trans-acting integration sequence comprises a transcription activator-like effector nuclease (TALEN). TALEN is a restriction enzyme that can be modified to cut specific sequences in DNA. Methods for using TALEN are described, for example, in WO2014134412A1, WO2013163628A2 and WO2014040370A1, which are incorporated by reference in their entirety.

[0422] In some embodiments, at least one trans-acting integration sequence comprises a non-RNA-guided nuclease and / or a fusion and / or derivative thereof. In some embodiments, insertion of the library construct into the insect cell genome is mediated by at least two trans-acting integration sequences, at least one trans-acting integration sequence comprises an RNA-guided nuclease and / or a fusion and / or derivative thereof, and at least one trans-acting integration sequence comprises a gRNA sequence.

[0423] In some embodiments, insertion of the library construct into the insect cell genome is mediated by at least one trans-acting integration sequence, the at least one trans-acting integration sequence comprising a nuclease (e.g., an RNA-guided nuclease or a non-RNA-guided nuclease) and / or fusions and / or derivatives thereof, and the library construct comprises at least one pair of cis-acting integration sequences that are homology arms. In some embodiments, the library construct comprises an identifier and / or payload between two viral packaging sequences (e.g., viral repeat sequences), all of which are located between cis-acting integration sequences that comprise homology arm sequences.

[0424] In some embodiments, insertion of the library construct into the insect cell genome is mediated by at least two trans-acting integration sequences, at least one of which comprises a nuclease (e.g., an RNA-guided nuclease) and / or a fusion and / or derivative thereof, and at least one of which comprises a gRNA sequence. In some embodiments, the library construct comprises an identifier and / or payload between two viral packaging sequences (e.g., viral repeat sequences), all of which are located between cis-acting integration sequences that comprise homology arm sequences.

[0425] In some embodiments, the library construct is integrated into the genome of insect cells by nuclease-mediated integration (for example, using CRISPR / Cas9). In some embodiments, the integration of the library construct is mediated by the cell's endogenous natural DNA repair mechanism. For example, integration can be performed simply by introducing exogenous DNA into cells, allowing site-specific nucleases to create integration sites, and then integrating donor DNA.

[0426] In some embodiments, insertion of the library construct into the insect cell genome is mediated by Cas9. In some embodiments, the library construct comprises an identifier and / or a payload between AAV ITR sequences, all of which are located between cis-acting integration sequences that include homology arm sequences.

[0427] Some exemplary scenarios for nuclease-mediated integration of library constructs are shown in Table 5 below. Each of the embodiments in Table 4 can relate to a single contiguous or discontinuous library construct. In some embodiments, the library construct is a discontinuous library construct, and individual constructs comprising identifiers are integrated into the genome. In some embodiments, the library construct is a discontinuous library construct, and two or more individual constructs of the library construct are integrated into the insect genome by nuclease-mediated integration. In some embodiments, all of the individual constructs of the discontinuous library construct are integrated into the insect genome by nuclease-mediated integration.

[0428] [Table 4]

[0429] Recombinase-mediated integration In some embodiments, the library construct is integrated into the insect genome by recombinase-mediated integration. For example, in some embodiments, a library construct is provided that includes a first set of viral repeat sequences flanking an identifier and / or a payload, the first set of viral repeat sequences flanking cis-acting integration sequences that include a recombinase recognition site.

[0430] In some embodiments, at least one trans-acting integration sequence comprises a recombinase, hi some embodiments, the recombinase comprises Cre, Flp, Dre, PhiC31, and / or Bxb1, and / or derivatives and / or fusions thereof.

[0431] In some embodiments, the trans-acting integration sequence comprises a recombinase including Cre. In some embodiments, Cre mediates integration of the library construct, or a portion thereof, into the insect cell genome. In some embodiments, the library construct comprises a first set of viral repeat sequences flanking the identifier and / or payload (e.g., AAV ITRs for packaging an AAV vector), wherein the first set of viral repeat sequences are flanked by cis-acting integration sequences comprising LoxP sites.

[0432] In some embodiments, the trans-acting integration sequence comprises a recombinase comprising Bxb1. In some embodiments, Bxb1 mediates integration of the library construct, or a portion thereof, into the insect cell genome. In some embodiments, the library construct comprises a first set of viral repeat sequences flanking the identifier and / or payload (e.g., AAV ITRs for packaging an AAV vector), wherein the first set of viral repeat sequences is flanked by cis-acting integration sequences comprising Att sites.

[0433] In some embodiments, the trans-acting integration sequence comprises a recombinase comprising Flp. In some embodiments, Flp mediates integration of the library construct, or a portion thereof, into the insect cell genome. In some embodiments, the library construct comprises a first set of viral repeat sequences flanking the identifier and / or payload (e.g., AAV ITRs for packaging an AAV vector), wherein the first set of viral repeat sequences is flanked by cis-acting integration sequences comprising Frt sites.

[0434] Some exemplary scenarios for recombinase-mediated integration of library constructs are shown in Table 6 below. Each of the embodiments in Table 5 can relate to a single contiguous or discontinuous library construct. In some embodiments, the library construct is a discontinuous library construct, and individual constructs containing identifiers are integrated into the genome. In some embodiments, the library construct is a discontinuous library construct, and two or more individual constructs of the library construct are integrated into the insect genome by recombinase-mediated integration. In some embodiments, all of the indivi...

Claims

1. a. Producing a plurality of adeno-associated virus (AAV) vectors from a library of insect cells, the library of insect cells comprising a plurality of insect cells, each insect cell being individually: (i) a nucleic acid sequence comprising a barcode disposed between two functional AAV ITR sequences; (ii) one or more library variants that provide one or more perturbations, wherein at least one library variant comprises a gRNA; (iii) an RNA-guided nuclease, and (iv) one or more nucleic acid sequences essential for the production of an AAV vector Including, producing, wherein the plurality of insect cells produces a plurality of AAV vectors, each AAV vector comprising an identifier corresponding to an identifier of the insect cell that produced it; b. detecting the barcodes of the plurality of AAV vectors; The method includes:

2. 1. A method for selecting insect cells for producing adeno-associated virus (AAV) vectors, the method comprising: a. transfecting a library of insect cells with one or more sequences essential for the production of said AAV vector, wherein each of said plurality of insect cells individually: (i) a nucleic acid sequence comprising a barcode disposed between two functional AAV ITR sequences; (ii) one or more library variants that provide one or more perturbations, wherein at least one library variant comprises a gRNA; and (iii) RNA-guided nuclease said transfecting comprising: b. Producing a plurality of AAV vectors from the library of insect cells, each of the AAV vectors comprising a barcode that corresponds to a barcode of the individual insect cell that produced it; c. detecting the barcodes of the plurality of AAV vectors, wherein the abundance of the barcodes correlates with the production and / or secretion of AAV vectors by the insect cells; The method comprising:

3. The method of claim 1 or 2, wherein the one or more sequences essential for production of the AAV vector are in the context of a baculovirus vector.

4. 4. The method of any one of claims 1 to 3, further comprising the step of measuring the relative amount of a particular barcode relative to all barcodes present in the plurality of AAV vectors produced from the library of insect cells.

5. 5. The method of any one of claims 1 to 4, wherein the one or more perturbations are associated with increased AAV production and / or AAV secretion compared to a reference insect cell lacking the one or more perturbations.

6. The method of any one of claims 1 to 5, wherein at least one library variant is integrated into the insect cell genome located between a pair of cis-acting integration sequences.

7. The method according to any one of claims 1 to 6, wherein one or two copies of the modified nucleic acid sequence are integrated into the insect cell genome.

8. The method of any one of claims 1 to 7, wherein at least one library variant is present episomally in the insect cell.

9. 9. The method of any one of claims 1 to 8, wherein the two functional AAV ITR sequences comprise a human AAV1 ITR, a human AAV2 ITR, a human AAV3b ITR, a human AAV4 ITR, a human AAV5 ITR, a human AAV6 ITR, a human AAV7 ITR, a human AAV8 ITR, a human AAV9 ITR, a human AAV10 ITR, a human AAV11 ITR, a human AAV12 ITR, or a human AAV13 ITR.

10. 9. The method of any one of claims 1 to 8, wherein the two functional AAV ITR sequences comprise a bovine AAV (b-AAV) ITR, a canine AAV (CAAV) ITR, a mouse AAV1 ITR, a caprine AAV ITR, a rat AAV ITR, or an avian AAV (AAAV) ITR.

11. 11. The method of any one of claims 1 to 10, wherein the one or more sequences essential for production of the AAV vector comprise (a) an AAV Rep gene, (b) an AAV Cap gene, (c) one or more AAV helper genes, or (d) a combination thereof.

12. 12. The method of claim 11, wherein the one or more sequences essential for production of an AAV vector comprise an AAV Cap gene encoding a human AAV1 capsid protein, a human AAV2 capsid protein, a human AAV3b capsid protein, a human AAV4 capsid protein, a human AAV5 capsid protein, a human AAV6 capsid protein, a human AAV7 capsid protein, a human AAV8 capsid protein, a human AAV9 capsid protein, a human AAV10 capsid protein, a human AAV11 capsid protein, a human AAV12 capsid protein, or a human AAV13 capsid protein.

13. The method of any one of claims 1 to 12, wherein the one or more perturbations comprise an insertion, deletion, substitution, conversion, epigenetic modification, and / or rearrangement of an endogenous genomic coding sequence.

14. 14. The method of any one of claims 1 to 13, wherein the one or more library variants comprise at least two library variants, wherein the at least two library variants comprise at least one unique gene, at least one unique ORF, at least one unique gRNA sequence, and / or at least one unique non-coding nucleic acid, or a combination and / or a plurality thereof.

15. The method of any one of claims 1 to 14, wherein the method further comprises single cell sequencing.

16. The method of any one of claims 1 to 15, wherein the library of insect cells comprises Sf21 cells, Sf9 cells, BTI-TN-5B1-4 (High Five) cells, S2 cells, D. Mel2 cells, or any derivative thereof.

17. 1. An insect cell, or a population of insect cells comprising a plurality of insect cells, each insect cell comprising: (i) a modified nucleic acid sequence comprising a barcode disposed between two functional AAV ITR sequences, said nucleic acid sequence being present as an episome in said insect cell; (ii) one or more library variants that provide one or more perturbations, wherein at least one library variant comprises a gRNA; and (iii) an RNA-guided nuclease; and (iv) one or more nucleic acid sequences essential for the production of an AAV vector; and Including, The insect cell or the population of insect cells, wherein the population of insect cells produces a plurality of AAV vectors, each AAV vector comprising a barcode that corresponds to the barcode of the insect cell from which it was produced.

18. 1. An insect cell, or a population of insect cells comprising a plurality of insect cells, each insect cell comprising: (i) a modified nucleic acid sequence comprising a barcode disposed between two functional AAV ITR sequences, said nucleic acid sequence being integrated into an insect cell genome disposed between a pair of cis-acting integration sequences; (ii) one or more library variants that provide one or more perturbations, wherein at least one library variant comprises a gRNA; (iii) an RNA-guided nuclease, and (iv) comprises one or more nucleic acid sequences essential for the production of an AAV vector; The insect cell or the population of insect cells, wherein the population of insect cells produces a plurality of AAV vectors, each AAV vector comprising a barcode that corresponds to the barcode of the insect cell that produced it.

19. 19. The insect cell or population of insect cells of claim 17 or 18, wherein the insect cells individually comprise Sf21 cells, Sf9 cells, BTI-TN-5B1-4 (High Five) cells, S2 cells, D. Mel2 cells, or any derivative thereof.

20. 20. The insect cell or insect cell population of any one of claims 17 to 19, wherein the AAV vector comprises a human AAV1 capsid protein, a human AAV2 capsid protein, a human AAV3b capsid protein, a human AAV4 capsid protein, a human AAV5 capsid protein, a human AAV6 capsid protein, a human AAV7 capsid protein, a human AAV8 capsid protein, a human AAV9 capsid protein, a human AAV10 capsid protein, a human AAV11 capsid protein, a human AAV12 capsid protein, or a human AAV13 capsid protein.

21. 20. The insect cell or population of insect cells of any one of claims 17 to 19, wherein the AAV vector comprises a human ancestral AAV capsid protein.

22. 22. The insect cell or insect cell population of any one of claims 17 to 21, wherein the viral vector comprises an AAV vector, the AAV vector comprising a first set of two viral repeat sequences comprising a pair of inverted terminal repeats (ITRs), and the ITRs are or comprise a human AAV1 ITR(s), human AAV2 ITR(s), human AAV3b ITR(s), human AAV4 ITR(s), human AAV5 ITR(s), human AAV6 ITR(s), human AAV7 ITR(s), human AAV8 ITR(s), human AAV9 ITR(s), human AAV10 ITR(s), human AAV11 ITR(s), human AAV12 ITR(s), or human AAV13 ITR(s).

23. 22. The insect cell or population of insect cells of any one of claims 17 to 21, wherein the AAV vector comprises bovine AAV (b-AAV) capsid protein, canine AAV (CAAV) capsid protein, mouse AAV1 capsid protein, goat AAV capsid protein, rat AAV capsid protein, or avian AAV (AAAV) capsid protein.

24. 24. The insect cell or population of insect cells of any one of claims 17 to 23, wherein the viral vector comprises an AAV vector, the AAV vector comprising a pair of ITRs, the ITRs being or comprising bovine AAV (b-AAV) ITR(s), canine AAV (CAAV) ITR(s), mouse AAV1 ITR(s), goat AAV ITR(s), rat AAV ITR(s), or avian AAV (AAAV) ITR(s).

25. The at least one polynucleotide comprising one or more nucleic acid sequences essential for the formation of a viral vector is a. AAV Rep gene, b. AAV Cap gene, c. one or more AAV helper genes, or d. Combinations of these 25. The insect cell or insect cell population according to any one of claims 17 to 24, comprising:

26. 26. The insect cell population of any one of claims 17 to 25, wherein the one or more perturbations are associated with increased AAV production and / or AAV secretion compared to a reference insect cell population lacking the one or more perturbations.

27. 27. The insect cell or insect cell population of any one of claims 17 to 26, which produces a population of AAV vectors comprising at least one improved characteristic compared to an AAV population produced by an insect cell lacking said one or more perturbations.

28. 28. The method of any one of claims 1 to 16, or the insect cell or insect cell population of any one of claims 17 to 27, wherein the at least one barcode comprises a sequence that is from about 5 to about 25 nucleotides.

29. 28. The method of any one of claims 1 to 16 or the insect cell or insect cell population of any one of claims 17 to 27, wherein the insect cells each individually comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 library variants.

30. The method of any one of claims 1 to 16 or the insect cell or insect cell population of any one of claims 17 to 27, wherein said insect cells each individually comprise up to 100 library variants.

Images