Methods for enhancing adeno-associated virus (AAV) production

Abstract

Compositions, systems, and methods for enhancing adenovirus L4 region 33K protein (Ad L4-33K) expression in an AAV producing cell are disclosed. For example, a system for AAV production including (a) a first nucleic acid molecule encoding an adenovirus L4-33K protein; and (b) a second nucleic acid molecule comprising adenovirus genes E2A, L4, E4, and VA RNA is described.

Description

METHODS FOR ENHANCING ADENO-ASSOCIATED VIRUS (AAV) PRODUCTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of EP Patent Application No. 24305146.3, filed January 26, 2024, US Provisional Application No. 63 / 644,013, filed May 8, 2024, and US Provisional Application No. 63 / 713,654, filed October 30, 2024, the contents of all of which are incorporated by reference in their entirety for all purposes.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety.Said XML copy is named 761081SA9-494PC.xml, created on January 24, 2025, and is 47,235 bytes in size.BACKGROUND

[0003] Adeno-associated virus (AAV) is a DNA parvovirus that infects humans and various other animal species. AAV contains a single stranded DNA genome encoding sets of replication (Rep) and capsid (Cap) proteins. Flanking the Rep and Cap open reading frames at the 5' and 3' ends are inverted terminal repeat sequences (ITRs), which act as origins of nucleic acid replication and as packaging signals for the virus. AAV infection, however, becomes latent and does not produce progeny virus in the absence of a helper virus.

[0004] An adenovirus (Ad) helper virus is a small non-enveloped virus. The Ad genome encodes ~39 genes, which are classified as either early or late depending on whether they are expressed before or after DNA replication. Helper functions for AAV expression are provided by early Ad transcription units encoding proteins E1A, E1 B, E2A, and E4, and the transcription unit VA RNA. The E1A gene product transcriptionally activates the AAV p5 promoter which controls expression of the Rep78 / 68 proteins, while the E1 B 55K protein complexes with the E4orf6 gene product (E4) to play a role in both AAV mRNA transport and inhibition of the cellular DNA damage response pathway. Both the E4orf6 and the E2A singlestranded DNA binding protein (DBP) support AAV DNA replication. The VA RNAs inhibit the activity of double-stranded RNA-activated kinase (PKR), thus promoting AAV protein synthesis by interfering with PKR-mediated shutdown of cellular translation which is a component of the host cell defense against viral infection. The VA RNA’s can also work in conjunction with the DBP to promote AAV capsid protein synthesis.

[0005] Major late gene products are encoded in the transcription units L1 to L5. The L4 region encodes: L4-100K, a translation enhancer protein that targets specific late mRNAs, protein pVIII, a structural protein of the viral capsid, L4-22K, required for viral DNA packaging into the empty capsid, and splicing factor L4-33K. The Adenovirus L4 22K / 33K gene products (also referred to as “L4-22 / 33K”) have been shown to be essential for amplification of the AAV rep and cap genes in the context of an AAV packaging cell line. The L4 region overlaps with the E2A promoter sequence that is a component of the adenoviral helper plasmid used in HEK293 / transfection-based production systems. The L4 22K protein is required for rAAV production in the transfection platform and that the 33K gene product also contributes to rAAV yield. The L4 22 / 33K proteins have been reported to play a role in various aspects of the adenoviral life cycle, including regulation of early and late gene expression, splicing of viral mRNAs and packaging of the viral genome.

[0006] AAV engineered for viral vector-mediated gene transfer is a valuable tool for studying gene functions and gene therapeutics. For example, recombinant forms of AAV (rAAV) have been developed as vectors by replacing all viral genes with a therapeutic transgene expression cassette, while retaining the ITRs required for vector packaging and DNA replication, but otherwise lacking viral genes in its DNA genome. The rAAV can cross the cell membrane and deliver its cargo into a cell, including into the nucleus of a transduced cell without integrating into the host genome and provide long-term gene expression.

[0007] Production of viral vectors remains labor intensive. There is a need in the art to improve the efficiency of rAAV production systems.SUMMARY

[0008] The present disclosure is based, at least in part, on the discovery of increased Rep / Cap splicing and rAAV production with supplementation of the adenovirus (Ad) L4-33K protein. Ad L4-33K is a virus-encoded alternative RNA splicing factor that activates splicing of viral late gene transcripts.

[0009] In one aspect, the disclosure provides a system for adeno-associated virus (AAV) production, the system comprising: (a) a first nucleic acid molecule encoding an adenovirus L4-33K protein; and (b) a second nucleic acid molecule comprising adenovirus genes E2A, L4, E4, and VA RNA.

[0010] In some embodiments, the first nucleic acid molecule is present in a first vector or first adenovirus and the second nucleic acid molecule is present in a second vector or second adenovirus; and the molar ratio of first vector or first helper adenovirus molecule to the second vector or second adenovirus is between 10:1 to 1 :10, between 5:1 and 1 :5, between 3:1 and 1 :3, between 2:1 and 1 :2; or between 1.5:1 and 1 :1.5; or the first nucleicacid molecule and second nucleic acid molecule are present in a single vector or a single adenovirus.

[0011] In some embodiments, the first nucleic acid molecule further comprises an EF1 A promoter.

[0012] In some embodiments, the adenovirus genes are wild type.

[0013] In some embodiments, the adenovirus genes are derived from a mutant virus. In some embodiments, the mutant virus is a replication defective mutant, a life cycle defective mutant, or a temperature sensitive mutant.

[0014] In one or more of the foregoing embodiments, the adenovirus (Ad) genes are serotype Ad2 genes or Ad5 genes.

[0015] In one or more of the foregoing embodiments, the system further comprises a third nucleic acid molecule encoding AAV replication (Rep) and AAV capsid (Cap) proteins. In some embodiments, the Rep protein serotype and the Cap protein serotype are the same. In some embodiments, the Rep protein serotype and the Cap protein serotype are different. In one or more of the foregoing embodiments, the third nucleic acid molecule further comprises a transgene flanked by inverted terminal repeat (ITR) sequences. In some embodiments, the first nucleic acid molecule is present in a first vector or first adenovirus and the third nucleic acid molecule is present in a third vector or third adenovirus, and the molar ratio of first vector or first adenovirus molecule to the third vector or third adenovirus is between 10:1 to 1:10, between 5:1 and 1:5, between 3:1 and 1 :3, between 2:1 and 1:2; or between 1.5:1 and 1:1.5. In some embodiments, the first nucleic acid and the second nucleic acid are present in a first vector or first adenovirus and the third nucleic acid is present in a third vector or a third adenovirus. In some embodiments, the system further comprises a fourth nucleic acid molecule nucleic acid molecule encoding transgene flanked by inverted terminal repeat (ITR) sequences.

[0016] In one or more of the foregoing embodiments, the second nucleic acid molecule further comprises an adenovirus pIX gene.

[0017] In one or more of the foregoing embodiments, the second nucleic acid molecule does not include one or more of an adenovirus E1A gene and an adenovirus E1B gene.

[0018] In another aspect, the disclosure provides an AAV producing cell line comprising the system of the above aspect and any of the foregoing embodiments.

[0019] In some embodiments, the cell line exhibits enhanced expression of the Adenovirus L4-33K protein as compared to a corresponding cell line comprising the adenovirus genes E2A, L4, E4, and VA RNA and not the first nucleic acid molecule encoding the L4-33K protein. In some embodiments, the cell line produces AAV with an enhanced titer in vector genomes per ml (vg / mL) as compared to the AAV titer of the corresponding cell line(vg / mL). In some embodiments, the enhanced titer is increased by 2-fold or greater as compared with the AAV titer of the corresponding cell line.

[0020] In another aspect, the disclosure provides an AAV production system comprising:(i) (a) a cell line having a genome stably integrated with one or more nucleic acid molecules encoding AAV Rep and AAV Cap proteins, an adenovirus L4-33K protein, and a transgene flanked by inverted terminal repeat (ITR) sequences; and (b) an adenovirus;(ii) (a) a cell line having a genome stably integrated with one or more nucleic acid molecules encoding AAV Rep and AAV Cap proteins, and a transgene flanked by inverted terminal repeat (ITR) sequences; and (b) an adenovirus encoding L4, E2A, E4 and VA RNA genes and a L4-33K protein; or(iii) (a) a cell line having a genome stably integrated with one or more nucleic acid molecules encoding AAV Rep and AAV Cap proteins, an adenovirus L4-33K protein, and a transgene flanked by inverted terminal repeat (ITR) sequences; and (b) an adenovirus encoding L4, E2A, E4 and VA RNA genes and a L4-33K protein.

[0021] In some embodiments, the adenovirus comprises L4, E2A, E4 and VA RNA genes.

[0022] In some embodiments, the adenovirus is wild type.

[0023] In some embodiments, the adenovirus is a mutant virus. In some embodiments, the mutant virus is a replication defective mutant, a life cycle defective mutant, or a temperature sensitive mutant.

[0024] In one or more of the foregoing embodiments, the adenovirus serotype is Ad2 or Ad5.

[0025] In one or more of the foregoing embodiments, the cell line is an insect cell line or a mammalian cell line. In some embodiments, the cell line is an Sf9 cell line. In some embodiments, the cell line is a CHO cell line, Vero cell line, HeLa cell line, MDCK cell line, BHK cell line, A549 cell line, amniocyte cell line, or HEK293 cell line. In some embodiments, the cell line is a HeLa S3 or HEK293 cell line.

[0026] In one or more of the foregoing embodiments, the cell line expresses an Adenovirus E1A protein and / or an Adenovirus E1B protein.

[0027] In one or more of the foregoing embodiments, the produced AAV is serotype AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, a variant thereof, or a pseudo-serotype AAV.

[0028] In another aspect, the disclosure provides a method for production of an adeno- associated virus (AAV), comprising: (a) integrating into a cell genome one or more nucleic acid molecules encoding AAV Rep and AAV Cap proteins, a transgene flanked by inverted terminal repeat (ITR) sequences; and an adenovirus L4-33K protein; (b) integrating into the cell genome adenovirus genes E2A, L4, E4, and VA RNA or infecting the cell genome withan adenovirus; (c) expanding the cell and producing the AAV within the cell; and (d) isolating the AAV.

[0029] In some embodiments, the cell is an insect cell or a mammalian cell. In some embodiments, the cell is an Sf9 cell. In some embodiments, the cell is a CHO cell, Vero cell, HeLa cell, MDCK cell, BHK cell, A549 cell, amniocyte, or HEK293 cell. In some embodiments, the cell is a HeLa S3 or HEK293 cell.

[0030] In one or more of the foregoing embodiments, the cell expresses an Adenovirus E1A protein and / or an Adenovirus E1B protein.

[0031] In one or more of the foregoing embodiments, the produced AAV is serotype AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, a variant thereof, or a pseudo-serotype AAV.

[0032] In one or more of the foregoing embodiments, whereby expression of the Adenovirus L4-33K protein is enhanced in the cell as compared to a corresponding cell comprising the adenovirus genes E2A, L4, E4, and VA RNA and not the nucleic acid molecule encoding the L4-33K protein. In some embodiments, the AAV titer is enhanced in vector genomes per ml (vg / mL) as compared to the AAV titer of the corresponding cell (vg / mL). In some embodiments, the enhanced AAV titer is increased by 2-fold or greater as compared to the AAV titer of the corresponding cell.

[0033] In another aspect, the disclosure provides an adeno-associated virus (AAV) production system comprising: (a) a cell; (b) a first vector comprising at least one nucleic acid molecule encoding an adenovirus L4-33K protein; (c) a second vector comprising one or more nucleic acid molecules encoding adenovirus genes E2A, L4, E4, and VA; and (d) a third vector comprising one or more nucleic acid molecules encoding AAV Rep and AAV Cap proteins and a transgene flanked by inverted terminal repeat (ITR) sequences; or a third vector comprising one or more nucleic acid molecules encoding AAV Rep and AAV Cap proteins and a fourth vector comprising a transgene flanked by ITR sequences.

[0034] In some embodiments, the cell is an insect cell or a mammalian cell.

[0035] In some embodiments, the cell is an Sf9 cell.

[0036] In some embodiments, the cell is a CHO cell, Vero cell, HeLa cell, MDCK cell,BHK cell, A549 cell, amniocyte, or HEK293 cell.

[0037] In some embodiments, the cell is a HeLa S3 or HEK293 cell.

[0038] In one or more of the foregoing embodiments, the cell expresses an adenovirus E1A protein and / or an adenovirus E1 B protein.

[0039] In another aspect, the disclosure provides a method of producing an adeno- associated virus (AAV), comprising:(a) transfecting a cell with two or more vectors comprising:(i) one or more nucleic acid molecules encoding an adenovirus L4-33K protein;(ii) one or more nucleic acid molecules encoding adenovirus genes E2A, L4, E4, and VA;(iii) one or more nucleic acid molecules encoding an AAV capsid (Cap) protein and one or more nucleic acid molecules encoding an AAV replication (Rep) protein; and(iv) one more nucleic acid molecules encoding a transgene flanked by inverted terminal repeat (ITR) sequences;(b) culturing the cell under conditions suitable for producing the AAV; and (c) isolating the AAV.

[0040] In some embodiments, either:(A) one of the two or more vectors comprises the one or more nucleic acid molecules encoding an AAV capsid (Cap) protein and one or more nucleic acid molecules encoding an AAV replication (Rep) protein; and the one more nucleic acid molecules encoding a transgene flanked by inverted terminal repeat (ITR) sequences;(B) one of the two or more vectors comprises the one or more nucleic acid molecules encoding adenovirus genes E2A, L4, E4, and VA, and the one more nucleic acid molecules encoding the transgene flanked by ITR sequences;(C) one of the two or more vectors comprises the one or more nucleic acid molecules encoding an AAV capsid (Cap) protein and one or more nucleic acid molecules encoding an AAV replication (Rep) protein; and the one or more nucleic acid molecules encoding adenovirus genes E2A, L4, E4, and VA;(D) one of the two or more vectors comprises the one or more nucleic acid molecules encoding an AAV capsid (Cap) protein and one or more nucleic acid molecules encoding an AAV replication (Rep) protein; and the one or more nucleic acid molecules encoding the adenovirus L4-33K protein; or(E) one of the two or more vectors comprises the one more nucleic acid molecules encoding a transgene flanked by inverted terminal repeat (ITR) sequences; and the one or more nucleic acid molecules encoding the adenovirus L4-33K protein.

[0041] In some embodiments, the two or more vectors comprise:(a) a first vector comprising the one or more nucleic acid molecules encoding the adenovirus L4-33K protein;(b) a second vector comprising the one or more nucleic acid molecules encoding adenovirus genes E2A, L4, E4, and VA;(c) a third vector comprising the one or more nucleic acid molecules encoding the AAV Cap protein and the one or more nucleic acid molecules encoding the AAV Rep protein, and optionally the one more nucleic acid molecules encoding the transgene flanked by ITR sequences.

[0042] In some embodiments, the two or more vectors comprise:(a) a first vector comprising the one or more nucleic acid molecules encoding the adenovirus L4-33K protein;(b) a second vector comprising the one or more nucleic acid molecules encoding adenovirus genes E2A, L4, E4, and VA;(c) a third vector comprising the one or more nucleic acid molecules encoding the AAV Cap protein and the one or more nucleic acid molecules encoding the AAV Rep protein, and(d) a fourth vector comprising the one more nucleic acid molecules encoding the transgene flanked by ITR sequences.

[0043] In some embodiments, the two or more vectors comprise:(a) a first vector comprising the one or more nucleic acid molecules encoding the adenovirus L4-33K protein;(b) a second vector comprising the one or more nucleic acid molecules encoding adenovirus genes E2A, L4, E4, and VA, and the one more nucleic acid molecules encoding the transgene flanked by ITR sequences; and(c) a third vector comprising the one or more nucleic acid molecules encoding the AAV Cap protein and the one or more nucleic acid molecules encoding the AAV Rep protein.

[0044] In some embodiments, the two or more vectors comprise:(a) a first vector comprising the one or more nucleic acid molecules encoding the adenovirus L4-33K protein;(b) a second vector comprising the one or more nucleic acid molecules encoding adenovirus genes E2A, L4, E4, and VA, and the one more nucleic acid molecules encoding the transgene flanked by ITR sequences;(c) a third vector comprising the one or more nucleic acid molecules encoding the AAV Cap protein and the one or more nucleic acid molecules encoding the AAV Rep protein.

[0045] In one or more of the foregoing embodiments, the two or more vectors further comprise one or more nucleic acid molecules encoding an adenovirus pIX gene.

[0046] In one or more of the foregoing embodiments, the two or more vectors do not include one or more of an adenovirus E1A gene and an adenovirus E1 B gene.

[0047] In one or more of the foregoing embodiments, the cell is an insect cell or a mammalian cell. In some embodiments, the cell is an Sf9 cell. In some embodiments, the cell is a CHO cell, Vero cell, HeLa cell, MDCK cell, BHK cell, A549 cell, amniocyte, or HEK293 cell. In some embodiments, the cell is a HeLa S3 or HEK293 cell.

[0048] In one or more of the foregoing embodiments, the cell expresses an adenovirus E1A protein and / or an adenovirus E1 B protein.

[0049] In one or more of the foregoing embodiments, the produced AAV is serotype AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, a variant thereof, or a pseudo-serotype AAV.

[0050] In one or more of the foregoing embodiments, expression of the L4-33K protein is enhanced in the cell as compared to a corresponding cell comprising the adenovirus genes E2A, L4, E4, and VA, which has not been transfected the one or more nucleic acid molecules encoding the adenovirus L4-33K protein. In some embodiments, the AAV titer is enhanced in vector genomes per ml (vg / mL) as compared to the AAV titer of the corresponding cell (vg / mL). In some embodiments, the enhanced AAV titer is increased by 2-fold or greater as compared to the AAV titer of the corresponding cell.BRIEF DESCRIPTION OF DRAWINGS

[0051] Figures 1A-D demonstrate results comparing wild type Adenovirus type 5 (wtAd5) and temperature sensitive variant Ad5ts149 for recombinant Adenovirus-associated viral vector (rAAV) and Adenovirus (Ad) replication and production in HeLa Producer Cell Line (PCL) (vector genome copies / mL for wtAd5-infected or Adts149-infected cells harvested at 48 hrs post infection (hpi)): AAV Productivity (A), AAV replication (B), Ad productivity (C), and Ad replication (D). Blank bar represents 37°C and Grey bar represents 39°C; DRG: DNase- resistant genome; TVG: Total viral genome; wt: wild type. The terms “adts149” and “ad5ts149” are used Interchangeably throughout this disclosure to refer to an adenoviral mutant harboring a thermosensitive mutation.

[0052] Figures 2A-E depict results demonstrating the effect of suppression of Ad replication and production with Adts149 at 39°C on Rep / Cap transcription, splicing and protein expression: (A) shows Western analysis of total cell lysates harvested at 48 hpi for Ad- infected PCLs using anti-Rep, anti-Cap, and anti-beta-tubulin antibodies; (B) Amplified Rep and Cap gene copies quantified via dPCR on extrachromosomal DNA from Ad-infected cells at 48hpi; (C) shows Northern blot analysis using a Cap-specific probe (left) and a schematic diagram of RT-dPCR amplicon and Northern blot probe location on rep / cap transcripts (right) (Diagram not drawn to scale. The illustration was created with BioRender.com. Filled rectangles represent open reading frames (ORFs). Short double arrow lines represent qPCR amplicons targeting all rep, rep / cap and spliced rep / cap transcripts generated from primer / probe sets listed in Table 1. The amplicon targeting splicing spans the major splice junction, and the double dashed line indicates the skipped intron. Long double arrow line represents northern blot probe target region within cap coding sequence. (D) Relative splicing percentage for Rep / Cap transcripts calculated from multiplex RT-dPCR on total RNA isolated for Ad infected cells at 48hpi; and (E) Relative Rep and Cap mRNA level were quantified from multiplex RT-dPCR using total RNA isolated at 48 hpi.

[0053] Figures 3A-N depict results demonstrating reduced Ad L4 gene expression at 39°C with adts149 via Ad gene expression profiling: Relative mRNA levels for E1A (A), E2A (C), HEXON (E), Ad L4 (G, H, I); Western blot analysis of total cell lysates harvested at 48 hpi for Ad-infected PCLs using anti-E1A (B), anti-Ad5 E2A (D), anti-Ad5 capsid (F) and anti-beta- tubulin antibodies; and Relative mRNA levels for E1 B55K (J), E1 B19K (K), E4orf6 / 7(L), VA RNA (M), and E2B (N). Relative mRNA were quantified from multiplex RT-dPCR using total RNA isolated at 48 hpi.

[0054] Figures 4A-G depict results demonstrating the effect of L4 gene expression knockdown on Rep / Cap transcription, splicing and expression: (A) shows a genomic schematic of Ad major late transcription unit illustrating the L4 mRNA transcripts and complementary positions for DsiRNA 100K A&B, 22 / 33K and L4 with predicted L4 expression level upon DsiRNA treatment listed below (Diagram not drawn to scale, adapted from Biasiotto et al. 2015 and Su et al. 2024, and created with BioRender); (B) and (C) demonstrates the knockdown efficiency for L4 targets (at least 90%); (D) shows knockdown of 22 / 33K reduced Rep / Cap transcription 100-fold; (E) shows knockdown of L4 genes decreased Rep / Cap splicing from 75% to around 50%; (F) provides Western blotting results showing Rep / Cap expression was significantly reduced when knocking down 22 / 33K; and (G) shows the AAV titer for each sample, normalized to the non-targeting siRNA negative control. The terms “22 / 33K” and “22K / 33K” are used interchangeably throughout this disclosure to refer to an adenovirus L4 region of the viral genome, which contains the coding sequences for 22K and 33K proteins.

[0055] Figures 5A-E depict results demonstrating the effect of L4 gene supplementation on Rep / Cap transcription and splicing in Adts149 at 39°C: (A) shows a schematic representation of L4 constructs expressing individual genes L4-100K, 22K, 33K or PVIII driven by the EF1a promoter using nucleofector 4-D; (B) shows the splicing percentage value for each condition; (C) shows relative Rep / Cap mRNA expression comparing different L4 gene supplementation to that of pUC57 control; (D) provides Western blotting results of Rep / Cap protein expression; and (E) shows the AAV titer for each sample, normalized to the pUC57 control.

[0056] Figures 6A-E depict results demonstrated the effect of Ad L4-33K supplementation on Rep / Cap splicing and rAAV productivity in triple transfected HEK293 cells: (A) shows a schematic representation co-transfection into HEK293 of pAAV with rep / cap / transgene, pAdhelper and EF1a promoter driven L4 construct or pUC57 were at molar ratio of 1 :1 :1 ; (B) shows the splicing percentage value for each condition; (C) shows the fold change value for Rep / Cap mRNA transcripts; (D) shows the AAV Titer for each sample, normalized to the pUC57 control; and (E) shows enhanced rAAV production when L4-33K supplementation was tested with three additional capsids / transgenes.

[0057] Figures 7A-D show results comparing wild type Adenovirus type 5 (wtAd5) and temperature sensitive variant Ad5ts149 for recombinant Adenovirus-associated viral vector (rAAV) and Adenovirus (Ad) replication and production in Producer Cell Line (PCL) (A model PCL was infected with wtAd5 and Ad5ts149 at the MOI indicated and harvested at 48 hpi for analyses. Relative adenovirus production is shown in (A), Ad replication is shown in (B), rAAV production is shown in (C), and rAAV replication is shown in (D), quantified and normalized to that of the wtAd5 MOI=5 infection at 37°C condition. Blank bars represent results from 37°C and grey bars represent results from 39°C infection. Data was shown as mean ± SD of three or four biological replicates and analyzed using two-way ANOVA followed by Sidak's test in Graphpad Prism 10.2.3. Data used for plotting is set forth in TABLE 2.

[0058] Figures 8A-L depict results showing suppression of adenovirus replication and production with Ad5ts149 at 39°C alters AAV Rep / Cap protein expression and alters AAV rep / cap transcription and splicing. (A) Representative AAV Cap Western blot images analyzed using total cell lysates prepared from cells harvested from experiments described in Figure 1. B-tubulin was used as the loading control. (B) Densitometric analysis of Cap VP3 expression in A, normalized to tubulin and presented as relative ratio to wtAd5 infection of MOI=5 at 37°C. (C) Representative Rep expression detected by Western blot. Densitometric analysis of Rep78 (D) and Rep68 (E) from C were presented as relative fold-change normalized to tubulin and then wtAd5 infection of MOI=5 at 37°C. Rep52 (F) and Rep40 (G) expression were analyzed using densitometry from western blot images of three independent biologic replicates. Expression was normalized to p-tubulin for each sample followed by normalization to that of wtAd5 MOI=5 37°C condition. Relative expression of Rep52 or Rep40 were shown as mean ± SD of three biological replicates and analyzed using two-way ANOVA followed by Sidak's test in Graphpad Prism 10.2.3. (H) Rep gene copies / cell quantified via dPCR using extrachromosomal DNA extracted from samples collected 48 hpi. (I) Northern analysis of rep / cap transcripts from total RNA isolated from 48 hpi samples. Northern blot probe locates in cap region but targets all 6 transcripts from p5, p19 and p40 promoters. Each of the AAV transcripts is labelled with the expected size in kb and u (unspliced) or s (spliced). UN: uninfected. Bottom: ethidium bromide-stained gel was used to demonstrate total RNA loading. Relative rep (J) and total rep+cap (K) mRNA levels were quantified by multiplex RT-dPCR using total RNA isolated from 48 hpi samples. As the producer cell line expression system has the wild type AAV rep and cap gene arrangement, rep and rep+cap transcripts were assessed and presented instead of rep and cap separately due to the use of the same polyA signal. (L) AAV rep / cap splicing percentage evaluated from multiplex RT-dPCR. Data was shown as mean ± SD of three biological replicates except two for wtAd5 100 lU / cell condition and analyzed using two-way ANOVA followed by Sidak’s test in Graphpad Prism 10.2.3.

[0059] Figures 9A-I depict results of Ad gene expression profiling showing reduced late gene but comparable or higher early gene expression at 39°C with Ad5ts149: (A, B) show relative mRNA levels for Ad early genes E1A (A) and E2A (B) at 48 hpi via RT-dPCR; (C, E) show representative adenovirus E1A.E2A and Ad late protein Western blot analysis at 48 hpi from adenovirus-infected PCL cells; B-tubulin was used as the loading control (Data was shown as mean ± SD of three biological replicates except two for wtAd5 1001 U / cell condition and analyzed using two-way ANOVA followed by Sidak's test in Graphpad Prism 10.2.3.) (D) show relative adenovirus hexon mRNA levels quantified via RT-dPCR; (F) shows an adenovirus genomic schematic illustrating major late transcription units with focus on the L4 mRNA transcripts and DsiRNA targeting positions for 100K A & B, 22 / 33K and L4A are annotated. Open reading frame (ORF) for each L4 gene product is shown as black rectangles. Predicted impacts on L4 expression level upon each DsiRNA transfection is listed below the genomic schematic. Diagram not drawn to scale. The illustration was adapted from Biasiotto et al. and Su et al. and created with BioRender.com; and (G, H, I) show relative mRNA levels for Ad L4 gene products quantified via RT-dPCR from total RNA isolated at 48 hpi and using qPCR primers described in F and Table 1. Data shown as mean ± SD of triple biological replicates except two biological replicates for wtAd5 100I U / cell condition and analyzed using two-way ANOVA followed by Sidak's test in Graphpad Prism 10.2.3.

[0060] Figures 10A-H depict results showing knockdown of adenovirus L4 gene expression reduces rep / cap transcription, splicing and expression. PCL cells were transfected with the indicated DsiRNAs followed by wtAd5 infection at 5 I U / cell immediately after the DsiRNA nucleofection. Analyses were performed with samples harvested at 72 hpi. (A) Relative adenovirus L4 gene expression evaluated by RT-dPCR. (B) Relative rAAV volumetric productivity from L4 DsiRNA compared to non-targeting DsiRNA negative control (NC). (C) Relative rep copies determined from extrachromosomal DNA with dPCR. (D) Relative total rep and cap mRNA level quantified from total RNA by multiplex RT-dPCR. (E) Top: Northern blot analyzing rep / cap transcripts with a probe located within cap sequence. Middle: Contrast- adjusted Northen Blot image to visualize weaker bands. Bottom: ethidium bromide-stained gel demonstrating total RNA loading. (F) Relative ratio of spliced 2.3s and unspliced 2.6u p40 transcripts from densitometric analysis of Northern blot in (E). (G) AAV splicing evaluated from multiplex RT-dPCR upon L4 gene knockdown via DsiRNA. (H) AAV Rep, Cap and adenovirus E2A protein expression analyzed by Western blot with B-tubulin as a loading control. PCL cells were transfected with the indicated DsiRNAs followed by wtAd5 infection at 5 I U / cell immediately after the DsiRNA nucleofection. Analyses were performed with samples harvested at 72 hpi. NC: Non-targeting negative DsiRNA control. Data was shown as mean ±SD of three biological replicates and analyzed by one-way ANOVA followed by Dunnett tests in Graphpad Prism 10.2.3.

[0061] Figures 11A-D depict results showing supplementation of adenovirus L4-33K improves rep / cap splicing, expression and AAV production in the context of Ad5ts149 infection at 39°C. (A) Relative rAAV titer normalized to the pUC57 control. (B) Relative total rep and cap mRNA levels evaluated with RT-dPCR analysis. The fold change was calculated compared to the pUC57 control. (C) AAV rep / cap splicing evaluated from multiplex RT-dPCR. (D) Representative AAV Rep and Cap protein expression in whole cell lysates analyzed via Western blot. AAV producer cells were transfected with L4 constructs expressing individual gene L4-100K, 22K, 33K or pVIII driven by the EF1a promoter. Transfected cells were infected at 24 hrs post-transfection with Ad5ts149 at 15 lll / cell and incubated at 39°C. Cells were harvested for downstream analysis at 48 hpi. B-tubulin was used as the loading control. Bar graph data was shown as mean ± SD of five biological replicates and analyzed by one-way ANOVA followed by Dunnett tests in Graphpad Prism 10.2.3.

[0062] Figures 12A-C depict results showing Ad L4 supplementation enhances AAV vector production in the context of the HEK293-transient transfection system: A plasmid containing rep / cap / transgene, pAdhelper and EF1a promoter driven L4 construct or pUC57 vehicle control were co-transfected into HEK293 cells at molar ratio of 1 :1 :1. Cultures were harvested 72 hrs post transfection for downstream analysis. Cap a / b / c / d refers to four different capsids including one natural serotype and three engineered capsid variants derived from different serotypes. Gene 1 / 2 / 3 / 4 refers to four different transgenes in the AAV vector; vector genome sizes range from approximately 3.7 kb to 4.6 kb. (A) shows relative rAAV titer from each L4 gene co-transfection normalized to the pUC57 control. Data was shown as mean ± SD of two or three biological replicates in Graphpad Prism 10.2.3. (B) shows AAV splicing analyzed from CapdGene4 transfection production as previously described. Data was shown as mean ± SD of three biological replicates and analyzed by one-way ANOVA followed by Dunnett tests in Graphpad Prism 10.2.3. (C) shows supplementing 33K via cloning into pAdhelper (pAdHelper- 33K) resulted in about 2-fold AAV productivity increase at TPP to pAdH ratio of 1 :1 for two different Triple-Play Plasmids (“TPP”s), TPP-1 and TPP-2 (1 :1 only was used for TPP-2).

[0063] Figures 13A-E show RT-dPCR analysis of adenovirus E1 B19K (A), E1 B55K (B), E4 (C), VA RNA (D) and E2B (E) mRNA expression level. Relative mRNA levels for E1 B19K, E1 B55K, E4, VA RNA and E2B were quantified from multiplex RT-dPCR using total RNA isolated at 48 hpi from each condition. Relative expression to GAPDH internal control and wtAd5 infection at 37°C at MOI=5 was calculated and presented. Data shown as mean ± SD of three biological replicates for all except two for the wtAd 100 lll / cell condition and analyzed using two-way ANOVA followed by Sidak's test in Graphpad Prism 10.2.3.

[0064] Figures 14A-B depict results demonstrating L4 gene knockdown efficiency: (A) Relative 100 / 22K mRNA and (B) relative 100K mRNA expression from L4 DsiRNA normalized to the non-targeting DsiRNA negative control (NC). Data was shown as mean ± SD of three biological replicates and analyzed by one-way ANOVA followed by Dunnett tests in Graphpad Prism 10.2.3.

[0065] Figure 15 depicts results demonstrating the impact of Ad L4 supplementation on rep / cap amplification in the context of an Ad5ts149 infection at 39°C: AAV rep DNA copy numbers were quantified via dPCR performed using extrachromosomal DNA extracted cells transfected with L4 constructs followed by Ad5ts149 infection. The fold change of amplified rep or cap copies / cell were compared to the pUC57 stuffer control. Data was shown as mean ± SD of three biological replicates and analyzed by one-way ANOVA followed by Dunnett tests in Graphpad Prism 10.2.3.

[0066] Figure 16 is a graphical representation of a producer cell line infected with the temperature sensitive mutant, Ad5ts149, or wtAd5, where infection with Ad5ts149 at the restrictive temperature results in reduced recombinant AAV titer and reduced splicing of the rep / cap transcripts. Optimization of Ad L4-33K expression facilitates expression and splicing of AAV rep / cap transcripts and represents a unique opportunity to optimize AAV vector production.

[0067] Figures 17A-D describe mRNA expression level of Ad L4 gene products upon supplementation in the context of Ad5ts149 infection at 39°C. (A) Expected RT-dPCR detection capability for transfected EF1a driven L4 constructs using Adenovirus L4 specific primer sets as illustrated in Figure 9F. Y: primer set able to detect expression; N: primer set not able to detect expression. (B-D) show Adenovirus L4 gene expression at 24hr, 48hr and 72hr post transfection was evaluated by RT-dPCR using L4 specific primer sets -100 / 22 / 33K (B), 100 / 22K (C), 100K (D) and normalized to GAPDH was presented as mean ± SD of two biological replicates. The horizontal dashed line represents L4 expression level achieved from wtAd5 at 39°C at 48 hpi.DETAILED DESCRIPTION

[0068] As described in the Background Section, a need exists to improve AAV production. Embodiments of the present disclosure relate generally compositions, systems, and methods for adeno-associated virus (AAV) production by supplementing or otherwise overexpressing adenovirus L4 region 33K protein (Ad L4-33K) in an AAV producing cell.

[0069] Before the subject matter of the present disclosure is described, it is to be understood that the subject matter is not limited to particular methods and experimental conditions described, as such methods and conditions can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting because the scope of the disclosure will be limited only by the appended claims.

[0070] The techniques and procedures described or referenced herein are described in Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds., 2003); the series Methods in Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds., 1995); Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (R. I. Freshney, 6th ed., J. Wiley and Sons, 2010); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., Academic Press, 1998); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., J. Wiley and Sons, 1993-8); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds., 1996); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Ausubel et al., eds., J. Wiley and Sons, 2002); Immunobiology (C. A. Janeway et al., 2004); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988- 1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V. T. DeVita et al., eds., J.B. Lippincott Company, 2011). All publications mentioned herein are incorporated herein by reference in their entirety.

[0071] As used herein, the term “vector” refers to any vehicle for the cloning of and / or transfer of a nucleic acid into a host cell. A vector can be a replicon to which another nucleic acid segment can be attached so as to bring about the replication of the attached segment. A “replicon” refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, i.e., capable of replication under its own control. The term “vector” includes both viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo, such as but not limited to a viral particle. A large number of vectors are known and used in the art including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of a polynucleotide into a suitable vector can be accomplished by ligating the appropriate polynucleotide fragments into a chosen vector that has complementary cohesive termini.

[0072] The term “plasmid” or “plasmid backbone” refers to an extrachromosomal circular DNA capable of autonomous replication in a given cell. A plasmid can include a selection gene in order to select or to identify a cell transfected therewith. Exemplary plasmids include but arenot limited to those derived from pBR322, plIC, pUCI9, pUC57, pJ24l, or pJ247, pBluescript, pREP4, pCEP4, and pCI. Plasmids can also be engineered by standard molecular biology techniques (Sambrook et ah, Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), N.Y.).

[0073] As used herein, the term “gene” refers to the segment of a DNA molecule that codes for a polypeptide chain (e.g., the coding region). In some embodiments, a gene can include regions involved in producing the polypeptide chain that are positioned immediately preceding, following, and / or intervening the coding region (e.g., regulatory elements such as a promoter, enhancer, polyadenylation sequence, 5'-untranslated region, 3'-untranslated region, or intron).

[0074] As used herein, the term “regulatory element” refers to one or more nucleic acid molecules, such as promoters, enhancers, terminators, polyadenylation sequences, introns, and the like, that provide for the expression of a coding nucleic acid molecule in a cell.

[0075] As used herein, the term “promoter element” refers to a nucleic acid sequence that assists with controlling expression of a coding nucleic acid molecule. A promoter element can be located 5' of the translation start site of a gene, or 3' of the coding nucleic acid molecule. In some embodiments, a promoter useful for gene therapy can be derived from a native gene of a target protein. In some embodiments, a promoter useful for gene therapy can be specific for expression in a particular cell or tissue of the target organism (e.g., a liver-specific promoter or muscle-specific promoter). Non-limiting examples of well-characterized promoter elements include the CMV early promoter, CMV enhancer / chicken p-actin promoter (CGA), the 3-actin promoter, chicken p-actin (CBA) promoters, and the methyl CpG binding protein 2 (MeCP2) promoter. A promoter can be a constitutive promoter or an inducible promoter.

[0076] As used herein, the term “polynucleotide” or “nucleic acid” refers to a polymeric molecule of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups (as can typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidates and thus can be an oligodeoxynucleoside phosphoramidate (P — NH2) or a mixed phosphoramidate-phosphodiester oligomer. In addition, a double-stranded polynucleotide can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.

[0077] Exemplary nucleic acids or polynucleotides of the disclosure include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), DNA-RNA hybrids, RNAi- inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a p-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino-a-LNA having a 2'- amino functionalization) or hybrids thereof.

[0078] The terms “polypeptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues can contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, a “polypeptide” refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native nucleic acid molecule, as long as the protein maintains the desired activity. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0079] As used herein, the term “naturally-occurring” as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide, or components thereof such as amino acids or nucleotides, that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.

[0080] As used herein, the term “derivative” refers to a nucleic acid, peptide, or protein (e.g., an AAV capsid protein) or a variant or analog thereof comprising one or more mutations and / or chemical modifications as compared to a corresponding full-length wild type nucleic acid, peptide or protein. Non-limiting examples of chemical modifications involving nucleic acids include, for example, modifications to the base moiety, sugar moiety, phosphate moiety, phosphate-sugar backbone, or a combination thereof. A nucleic acid molecule that encodes mutant gene constructs that can be useful with the plasmid system described herein can be identical to a wild type (i.e., unmutated) nucleic acid molecule or can be a different coding nucleic acid molecule, which nucleic acid molecule, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptides as the wild type coding nucleic acid molecule. One of ordinary skill in the art will recognize that each codon in a nucleic acid molecule (except AUG, which is ordinarily the only codon for methionine, and TGG, whichis ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each variation of a nucleic acid which encodes a same polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual gene therapy constructs.

[0081] One of ordinary skill in the art will recognize that individual substitutions, deletions or additions to a nucleic acid molecule that alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded nucleic acid molecule is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure. Conservative amino acid substitutions providing functionally similar amino acids are well known in the art. Dependent on the functionality of the particular amino acid, e.g., catalytic, structural, or sterically important amino acids, different groupings of amino acid can be considered conservative substitutions for each other.

[0082] As used herein, the term “percent (%) sequence identity” with respect to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in the reference polypeptide or nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid or nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software programs, for example, those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987), Supp. 30, section 7.7.18, Table 7.7.1 , and including BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. An example of an alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, theamino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. For purposes herein, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows: 100 times the fraction W / Z, where W is the number of nucleotides scored as identical matches by the sequence alignment program in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.

[0083] As used herein, the term “isolated” when referring to a molecule (e.g., nucleic acid or protein) or cell means the molecule or cell has been identified and separated and / or recovered from a component of its natural environment.

[0084] As used herein, the term “helper virus” for AAV refers to a virus that allows AAV to be replicated and packaged by a host cell. A number of helper viruses are known including adenoviruses, herpesviruses, baculovirus, and poxviruses such as vaccinia. Adenoviruses encompass a number of different subgroups. Numerous adenoviruses of human, non-human mammalian and avian origin are known and are readily available (e.g., ATCC). Viruses of the herpes family include, for example, herpes simplex viruses (HSV), Epstein-Barr viruses (EBV), cytomegaloviruses (CMV), and pseudorabies viruses (PRV).

[0085] As used herein, the term “recombinant viral vector” refers to a recombinant polynucleotide vector comprising one or more heterologous nucleic acid molecules (i.e., nucleic acid molecule not of viral origin). In the case of recombinant AAV vectors, the recombinant nucleic acid molecule is flanked by at least one inverted terminal repeat sequence (ITR). In some embodiments, the recombinant nucleic acid molecule is flanked by two ITRs.

[0086] As used herein, the term “recombinant AAV vector (rAAV vector)” refers to a polynucleotide vector comprising one or more heterologous nucleic acid molecules (i.e., nucleic acid molecules not of AAV origin) that are flanked by at least one AAV inverted terminal repeat sequence (ITR). Such rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV Rep and Cap gene products (i.e., AAV Rep and Cap proteins). When a rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection), then a rAAV vector can be referred to as a “pro-vector” which can be “rescued” by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. A rAAV vector can be in any of a number of forms, including, but not limitedto, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes, and encapsidated in a viral particle, e.g., an AAV particle. A rAAV vector can be packaged into an AAV virus capsid to generate a “recombinant adeno-associated viral particle (rAAV particle)”. “Recombinant AAV” (rAAV) and “AAV” are used interchangeably throughout the present disclosure.

[0087] As used herein, the term “rAAV virus” or “rAAV viral particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated rAAV vector genome.

[0088] As used herein, the term “heterologous” means derived from a genotypically distinct entity from that of the rest of the entity to which it is compared or into which it is introduced or incorporated. As it relates to nucleic acid molecules, such as coding sequences and / or control sequences, the term denotes nucleic acid molecules that are not normally joined together and / or are not normally associated with a particular cell. A cellular sequence (e.g., a gene or portion thereof) that is incorporated into a viral vector is a heterologous nucleotide acid molecule with respect to the vector. Thus, a “heterologous” nucleic acid molecule can be a nucleic acid molecule from an organism other than AAV or which is synthetically derived.

[0089] “Operably-linked” refers to the association of two or more elements of a nucleic acid molecule that are physically linked so that the function of one of the elements is affected by another. For example, a regulatory DNA element is said to be “operably linked to” or “associated with” a DNA element that codes for an RNA or a polypeptide if the two elements are situated such that the regulatory DNA element affects expression of the coding DNA element (i.e., that the coding nucleic acid molecule or functional RNA is under the transcriptional control of the promoter). Coding nucleic acid molecule can be operably-linked to regulatory elements in sense or antisense orientation.

[0090] As used herein, the term “transgene” refers to a polynucleotide that is introduced into a cell and is capable of being transcribed into RNA and optionally, translated and / or expressed under appropriate conditions. A transgene can confer a desired property to a cell into which it was introduced, or otherwise leads to a desired therapeutic or diagnostic outcome. In another aspect, it can be transcribed into a molecule that mediates RNA interference, such as miRNA, siRNA, or shRNA. A transgene can be as few as a couple of nucleotides or at least about 50, 100, 150, 200, 250, 300, 350, 400, 500, 1 ,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 7,500 nucleotides long or longer. A transgene can be for example, a viral genome. A transgene can be coding or non-coding nucleic acid molecule, or a combination thereof. A transgene can include one or more regulatory elements whereby transgene expression can be controlled.

[0091] As used herein, the terms “genome particles (gp)” or “genome copies” as used in reference to a viral titer, refer to the number of virions containing the recombinant AAV DNA genome, regardless of infectivity or functionality. The number of genome particles in aparticular vector preparation can be measured by procedures such as described in the Examples herein, or for example, in Clark et al. (1999) Hum. Gene Then, 10: 1031-1039; Veldwijk et al. (2002) Mol. Then, 6:272-278.

[0092] As used herein, the term “vector genome (vg)” can refer to one or more polynucleotides comprising a set of the polynucleotide molecules of a vector, e.g., a viral vector. A vector genome can be encapsidated in a viral particle. Depending on the particular viral vector, a vector genome can comprise single-stranded DNA, double-stranded DNA, or single-stranded RNA, or double-stranded RNA. A vector genome can include endogenous nucleic acid molecules associated with a particular viral vector and / or any heterologous nucleic acid molecules inserted into a particular viral vector through recombinant techniques. For example, a recombinant AAV vector genome can include at least one ITR sequence flanking a promoter, a stuffer, a nucleic acid molecule of interest (e.g., an RNAi), and a polyadenylation sequence. A complete vector genome can include a complete set of the polynucleotide molecules of a vector. In some embodiments, the nucleic acid titer of a viral vector can be measured in terms of vector genome (vg / mL). Methods suitable for measuring this titer are known in the art (e.g., quantitative PCR).

[0093] As used herein, the term “inverted terminal repeat” or “ITR” sequence refers to relatively short sequences found at the termini of viral genomes which are in opposite orientation.

[0094] As used herein, the term “AAV inverted terminal repeat (ITR)” sequence refers to an approximately 145-nucleotide sequence that is present at both termini of the native singlestranded AAV genome. The outermost 125 nucleotides of the ITR can be present in either of two alternative orientations, leading to heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contains several shorter regions of self-complementarity (designated A, A, B, B', C, C and D regions), allowing intra-strand base-pairing to occur within this portion of the ITR.

[0095] As used herein, the term “terminal resolution sequence” or “trs” refers to a sequence in the D region of the AAV ITR that is cleaved by AAV rep proteins during viral DNA replication. A mutant terminal resolution sequence is refractory to cleavage by AAV rep proteins.

[0096] As used herein, the term “effective amount” is an amount sufficient to effect beneficial or desired results, including clinical results (e.g., amelioration of symptoms, achievement of clinical endpoints, and the like). An effective amount can be administered in one or more administrations. In terms of a disease state, an effective amount is an amount sufficient to ameliorate, stabilize, or delay development of a disease.

[0097] As used herein, the terms “subject”, “patient”, “individual”, and “animal” are used interchangeably and refer to mammals, including, without limitation, human and veterinaryanimals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models. In certain embodiments, the individual or subject is a human.

[0098] As used herein, the term “treatment” refers to an approach for obtaining beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (e.g., not worsening) state of disease, preventing spread (e.g., metastasis) of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. The term “treat” is the verb form of “treatment”.

[0099] As used herein, the term “prophylactic treatment” refers to treatment, wherein an individual is known or suspected to have or be at risk for having a disorder but has displayed no symptoms or minimal symptoms of the disorder. An individual undergoing prophylactic treatment can be treated prior to onset of symptoms.

[0100] As used herein, a “therapeutic” agent (e.g., a therapeutic polypeptide, nucleic acid, or transgene) is one that provides a beneficial or desired clinical result. As such, a therapeutic agent can be used in a treatment as described above.

[0101] Ranges can be expressed herein as from “about” or “approximately” or “substantially” one particular value and / or to “about” or “approximately” or “substantially” another particular value. When such a range is expressed, other exemplary embodiments include from the one particular value and / or to the other particular value. Further, the term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e. , the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to ±10%, up to ±5%, and up to ±1 % of a given value. For example, as used herein, the expression “about 100” includes 99 and 101 and all values in between (e.g., 99.1 , 99.2, 99.3, 99.4, etc.) Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, or within 2-fold of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value.NUCLEIC ACID MOLECULES ENCODING A L4-33K PROTEIN

[0102] Materials and methods of the present disclosure feature nucleic acid molecules encoding an Adenovirus (Ad) L4-33K protein or a homolog thereof (i.e., an L4-33K-encoding nucleic acid molecule). In some embodiments, a L4-33K protein (also referred to as “33K protein” herein) can comprise or consist of an amino acid sequence as set forth in SEQ IDNO: 3, or a functional fragment or variant thereof. In some embodiments, a L4-33K protein can comprise or consist of an amino acid sequence as set forth in NCBI Reference Sequence: AP_000215.1 , or a functional fragment or variant thereof. In some embodiments, a L4-33K protein can comprise or consist of an amino acid sequence as set forth in NCBI Reference Sequence: AP_000179.1 , or a functional fragment or variant thereof. In certain embodiments, an L4-33K-encoding nucleic acid molecule can be a 33K gene derived from the L4 region of a helper Ad genome. An L4-33K-encoding nucleic acid molecule can be obtained from any helper Ad type, such as type 5 (Ad5) or type 2 (Ad2). An L4-33K-encoding nucleic acid molecule can include a wild type or codon optimized sequence for a desired expression system. In certain embodiments, an L4-33K-encoding nucleic acid molecule can include an Ad L4 region or fragment thereof. In certain embodiments, an L4-33K-encoding nucleic acid molecule can include nucleic acid molecules encoding one or more other L4 region proteins, such as an L4-100K protein (e.g., a “100k” protein having the amino acid sequence set forth in NCBI Reference Sequence: AP_000178.1 or AP_000214.1), an L4-22K protein (e.g., a “22k” protein having an amino acid sequence as set forth in SEQ ID NO: 6 or NCBI Reference Sequence: AP_000180.1 , or a functional fragment or variant thereof)), an L4-pVI II protein (e.g., a “pVIH” protein having an amino acid sequence as set forth in NCBI Reference Sequence: AP_000217.1 or AP_000181.1 , or a functional fragment or variant thereof. For example, an L4-33K-encoding nucleic acid molecule can include a sequence set forth in SEQ ID NO: 2 and / or SEQ ID NO: 5) In certain embodiments, an L4-33K-encoding nucleic acid molecule can limit or prevent alternative splicing of an L4 region sequence for expression of an Ad L4-22K protein (e.g., a 22K protein having an amino acid sequence as set forth in SEQ ID NO: 6).

[0103] In certain embodiments, an L4-33K-encoding nucleic acid molecule comprises, consists of, or consists essentially of a nucleic acid sequence having at least about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 2 or a functional fragment or derivative thereof. In certain embodiments, an L4-33K-encoding nucleic acid molecule comprises, consists of, or consists essentially of SEQ ID NO: 2, or a functional fragment or derivative thereof.

[0104] In certain embodiments, an L4-33K-encoding nucleic acid molecule can be present in a helper Ad genome and / or a helper Ad viral particle. In certain embodiments, an L4-33K- encoding nucleic acid molecule can be stably integrated into a genome of a cell line for AAV production.

[0105] An L4-33K-encoding nucleic acid molecule can be an isolated molecule or inserted into an expression cassette, vector, or plasmid as described above. In certain embodiments, an expression cassette comprising an L4-33K-encoding nucleic acid molecule can have a sequence as set forth in SEQ ID NO: 1. An L4-33K-encoding nucleic acid molecule can beoperatively linked to one or more regulatory sequences, including promoters, enhancer, polyadenylation signals and other expression elements, which influence expression magnitude and stability. For example, an L4-33K-encoding nucleic acid molecule can be operatively linked to a constitutive promoter that can drive high levels of expression, such as CMV or EF1A promoter. In certain examples, a L4-33K-encoding nucleic acid molecule can be operatively linked to an EF1A promoter. An EF1A promoter can have a sequence as set forth in SEQ ID NO: 7, for example.

[0106] In an aspect, a L4-33K encoding nucleic acid molecule can be present in addition to an Ad helper virus or in addition to Ad helper virus nucleic acid molecules (e.g., E2A, L4, E4, and VA RNA). That is, a L4-33K encoding nucleic acid molecule can be present in the compositions and systems described herein in addition to an adenovirus or helper nucleic acid molecule(s). In some aspects, a L4-33K encoding nucleic acid molecule can be present in addition to an adenovirus or in addition to one or more nucleic acid molecules comprising an L4 region of an adenovirus genome. In some embodiments, an adenovirus L4 region can have a nucleic acid sequence as described in NCBI Reference Sequence: AC_000007.1 CDS: 24108-27898 (Human adenovirus 2, complete genome) or AC_000008.1 CDS: 24061-27857 (Human adenovirus 5, complete genome), encoding transcription units that are translated into L4-100K, L4-22K, L4-33K and pVIII proteins.HELPER NUCLEIC ACID MOLECULES

[0107] Materials and methods of the present disclosure can include one or more helper nucleic acid molecules for AAV replication and packaging. A helper nucleic acid molecule can include helper virus sequences necessary for AAV replication, such as an adenovirus (Ad) or herpesvirus vector.

[0108] In certain embodiments, helper nucleic acid molecules includes Ad nucleic acid molecules. A helper nucleic acid can include one or more of Ad genes E1 A, E1 B, E2, E3, E4, encoding proteins IX and IVa2, regions L1-L5, and virus-associated (VA) RNAI and VA RNAII. Ad helper nucleic acid molecules can be derived from any Ad type, such as Ad5 or Ad2. In certain embodiments, an Ad nucleic acid molecule comprises Ad5 genes.

[0109] In certain embodiments, a helper nucleic acid molecule can encode one or more of Ad E4, L4, E2A, VA RNA, or fragments thereof. Adenovirus genes can be present in a vector, in an adenovirus, or integrated into a cell genome. In certain embodiments, Ad nucleic acid molecules can include, without limitation, Ad gene sequences for E2A, L4, E4 (orf6), and / or a VA RNA gene, or fragments thereof. In certain embodiments, Ad nucleic acid molecules can include VA RNA, L4, E4, and E2A genes, or fragments thereof. In certain embodiments, Ad nucleic acid molecules can include E2A, L4, E4 ORFs 1 , 2, 3, 4, and 6 / 7, and VA RNA genes, or fragments thereof. In certain embodiments, Ad nucleic acid molecules can include E2A, L4, E4 ORFs 1 , 2, 3, 4, and 6 / 7, and VA RNA genes, or fragments thereof. In certain embodiments,Ad nucleic acid molecules can include E2A, L4-100K, L4-33K, L4-22K, E4 ORFs 1 , 2, 3, 4, and 6 / 7, and VA RNA genes, or fragments thereof. In certain embodiments, Ad nucleic acid molecules can include E2A, L4-100K, L4-33K, L4-22K, pVIII, E4 ORFs 1 , 2, 3, 4, and 6 / 7, and VA RNA genes, as well as genes encoding other Ad gene products, or fragments thereof. In certain embodiments, an Ad nucleic acid molecule does not include an Ad E1A or E1 B gene.

[0110] In certain embodiments, Ad nucleic acid molecules can be present in an Ad genome and / or encapsidated in an Ad vector.

[0111] An Ad nucleic acid molecule can be an isolated molecule or inserted into one or more expression cassettes, vectors or plasmids as described above. In certain embodiments, Ad nucleic acid molecules can be inserted into a plasmid backbone, e.g., a pUC57 plasmid backbone. In certain embodiments, Ad nucleic acid molecules and a L4-33K-encoding nucleic acid molecule can be present on a single construct (e.g., plasmid backbone). In certain embodiments, Ad nucleic acid molecules can be stably integrated into a genome of a cell line for AAV production.NUCLEIC ACID MOLECULES ENCODING AAV REP / CAP PROTEINS

[0112] Materials and methods of the present disclosure can include one or more nucleic acid molecules encoding AAV non-structural proteins that mediate viral replication and the production of new virus particles (“Rep coding molecules”) and AAV structural proteins that form a functional AAV capsid (“Cap coding molecules”).

[0113] A Rep coding nucleic acid molecule can encode one or more AAV Rep proteins. In certain embodiments, a Rep coding nucleic acid molecule encodes Rep proteins that are necessary for viral genome replication and packaging into new virions. In certain embodiments, a Rep coding nucleic acid molecule can encode one or more of AAV Rep78, Rep68, Rep52, and Rep40. In certain embodiments, a Rep coding nucleic acid molecule can encode at least Rep78 or Rep68 and Rep52 or Rep40. In certain embodiments, a Rep coding nucleic acid molecule can encode Rep78 and Rep52 and / or Rep40 protein or Rep68 and Rep52 and / or Rep40. In certain embodiments, a Rep coding nucleic acid molecule can encode Rep78, Rep68, Rep52 and Rep40 proteins.

[0114] A Rep coding nucleic acid molecule can include a wild type or synthetic nucleic acid sequence encoding one or more AAV Rep proteins. A Rep coding nucleic acid molecule can be derived from any AAV, including but not limited to serotypes AAV1 , AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , or AAV13, or any other AAV, such as a chimeric AAV or a variant AAV. A Rep molecule can include a wild type sequence modified by insertion, deletion, truncation and / or missense mutations.

[0115] In certain embodiments, a Rep coding nucleic acid molecule can be present in an Ad genome, encapsidated in an Ad vector or hybrid herpes simplex virus type I (HSV-1) vector.In certain embodiments, a Rep coding nucleic acid molecule can be inserted into one or more expression cassettes, vectors or plasmids as described above. In certain embodiments, Ad nucleic acid molecules and a Rep coding nucleic acid molecule can be present on a single construct (e.g., plasmid backbone). In certain embodiments, a Rep coding nucleic acid molecule can be stably integrated into a genome of a cell line for AAV production.

[0116] A Cap coding nucleic acid molecule can encode one or more AAV Cap proteins. A Cap coding nucleic acid molecule can encode one or more AAV capsid subunits. In certain embodiments, a Cap coding nucleic acid molecule encodes a sufficient number of capsid subunits for producing a functional AAV capsid. AAV capsid structure is described in BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott- Raven Publishers).

[0117] A Cap coding nucleic acid molecule can include a wild type or synthetic nucleic acid molecule encoding one or more AAV Cap proteins. In certain embodiments, a Cap coding nucleic acid molecule can include one or more of VP1 , VP2, and VP3. A Cap coding nucleic acid molecule can be derived from any AAV, including but not limited to serotypes AAV1 , AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , or AAV13, or any other AAV, such as a chimeric AAV or a variant AAV. A Cap coding nucleic acid molecule can include a wild type sequence modified by insertion, deletion, truncation and / or missense mutations. A Cap coding nucleic acid molecule can be modified to provide an altered tropism. In certain embodiments, a Cap coding nucleic acid molecule encodes a capsid protein for transducing a specific cell, e.g., CNS, heart, liver, lung, pancreas, photoreceptor cells, retinal pigment epithelium, and / or skeletal muscle.

[0118] In certain embodiments, a Cap coding nucleic acid molecule can be present in a helper Ad genome and / or an Ad vector. In certain embodiments, a Cap coding nucleic acid molecule can be encapsidated in an Ad vector or HSV-1 vector. In certain embodiments, a Cap coding nucleic acid molecule can be inserted into one or more expression cassettes, vectors, or plasmids as described above. In certain embodiments, a Rep coding nucleic acid molecule and a Cap coding nucleic acid molecule can be present on a single construct (e.g., plasmid backbone). In certain embodiments, a Rep coding nucleic acid molecule and a Cap coding nucleic acid molecule can be encapsidated in an Ad vector or HSV-1 vector. A Cap coding nucleic acid molecule and a Rep coding nucleic acid molecule can encode proteins from the same AAV type, or from different AAV types. In certain embodiments, a Rep coding molecule, a Cap coding nucleic acid molecule and Ad nucleic acid molecules can be present on a single vector or a single expression cassette. In certain embodiments, a Cap coding nucleic acid molecule can be stably integrated into a genome of a cell line for AAV production.

[0119] In certain embodiments, Rep coding nucleic acid molecule and a Cap coding nucleic acid molecule can be present on a single Rep-Cap plasmid. A Rep-Cap plasmid can includean AAV promoter to control expression of AAV Rep and Cap proteins described above. A promoter can be any desired promoter, selected based on known considerations, such as the level of expression of a nucleic acid molecule functionally linked to the promoter and the cell type in which the vector is to be used. A promoter can be tissue / cell-specific. A promoter can be a prokaryotic, eukaryotic, fungal, nuclear, mitochondrial, viral, or plant promoter. A promoter can be exogenous or endogenous to the cell type being transduced by a vector. Promoters can be selected from, for example, bacterial promoters, known strong promoters such as SV40 or the inducible metallothionein promoter. A promoter can be a promoter of any AAV serotypes (e.g., an AAV P5 promoter). A promoter can be a chimeric regulatory promoter for targeted gene expression or promoter derived from actin genes, immunoglobulin genes, cytomegalovirus (CMV), adenovirus, bovine papilloma virus, adenoviral promoters, such as the adenoviral major late promoter, an inducible heat shock promoter, respiratory syncytial virus, Rous sarcomas virus (RSV), and the like. Non-limiting examples of suitable plasmid backbones for a Rep-Cap plasmid can include pHLPI9, pETCI8, pETCI9, and pAAV-RC2.AAV CONSTRUCTS

[0120] Materials and methods of the present disclosure can include one or more nucleic acid molecules encoding two AAV ITR sequences, which are located 5' and 3' to a polynucleotide sequence. The AAV ITR sequences can include any cis elements required for packaging allowing for production of recombinant AAV.

[0121] An AAV construct can include ITR sequences from any AAV serotype. Nucleic acid molecules of AAV ITR regions are known. An ITR can have a wild type nucleic acid sequence, or a modified sequence, e.g., by the insertion, deletion or substitution of nucleotides. An AAV ITR can be derived from any AAV serotype, including without limitation, AAV1 , AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , or AAV13, and 5' and 3' ITRs in an AAV vector can be independently selected therefrom.

[0122] An AAV construct can include a polynucleotide of any length positioned within two AAV ITR sequences (i.e., a polynucleotide can be flanked by ITR sequences). A polynucleotide can be a transgene of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000 or at least 10000 nucleotides. In certain embodiments, an ITR flanked polynucleotide sequence does not exhibit biological activity. For example, enhancers, promoters, splicing regulators, noncoding RNAs, antisense sequences, and / or coding sequences can be absent from a polynucleotide sequence positioned within two AAV ITR sequences. In certain embodiments, each of enhancers, promoters, splicing regulators,noncoding RNAs, antisense sequences, and coding sequences are absent. In certain embodiments, a polynucleotide sequence positioned within two AAV ITR sequences does not include an open reading frame.AAV VECTORS AND EXPRESSION CASSETTES

[0123] Materials and methods of the present disclosure can include an AAV vectors or expression cassettes obtained by cloning at least one heterologous nucleic acid molecule of interest (i.e., a transgene) into an AAV construct. For example, an AAV vector or expression cassette can include recombinant DNA containing a heterologous nucleic acid molecule, such as a transgene, flanked by ITR sequences for packaging into an AAV capsid. An AAV vector or expression cassette can be introduced into a eukaryotic cell line for producing AAV.

[0124] An AAV vector or expression cassette can be constructed using techniques for providing operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the DNA of interest, and a transcriptional termination region. The control elements can be selected to be functional in a mammalian cell. Termination signals, such as polyadenylation sites, can be included in the plasmid.

[0125] A heterologous nucleic acid molecule of an AAV vector or expression cassette can include a polynucleotide with biological activity of an anti-sense RNA molecule, shRNA, miRNA, a ribozyme, or a gene encoding a polypeptide of interest, and optionally one or more a nucleic acid sequences capable of directing expression of a particular heterologous nucleic acid sequence in an appropriate host cell (e.g., mammal). Non-limiting of nucleic acid sequences capable of directing expression include a promoter and termination signal. In certain embodiments, a heterologous nucleic acid molecule can be included in a chimeric expression cassette. In certain embodiments, an expression cassette can be naturally occurring, optionally obtained in a recombinant form for heterologous expression.

[0126] In certain embodiments, a heterologous nucleic acid molecule can be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000 nucleotides, at least 5000 nucleotides, at least 6000 nucleotides, at least 7000 nucleotides, at least 7500 nucleotides, or at least 8000 nucleotides.

[0127] In certain embodiments, a heterologous nucleic acid molecule (e.g., a transgene) can encode a polypeptide such as, but not limited to, a clotting factor, an enzyme, an antibody or other polypeptide of interest. In certain embodiments, a heterologous nucleic acid molecule can encode an RNA having a structural or therapeutic function such as, but not limited to, an antisense, siRNA, shRNA, miRNA, EGSs, gRNA, sgRNA, ribozyme, or aptamer.

[0128] In certain embodiments, a heterologous nucleic acid molecule can encode a peptide, polypeptide, or protein that binds to a specific target of interest, which can be useful for the treatment or prevention of disease in a subject. Examples of such heterologous nucleic acid molecules and associated peptides, polypeptides, or proteins include, but are not limited to, a gene encoding antibodies, MHC molecules, T-cell receptors, B-cell receptors, aptamers, avimers, receptor-binding ligands, or targeting peptides. Antibodies useful in the present disclosure can be monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, and any other modified configuration of a immunoglobulin molecule including an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies and specific binding fragments thereof can be murine, rat, human, or of any other origin (including chimeric or humanized antibodies). An antibody can include an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), or an unspecified class.

[0129] A heterologous nucleic acid molecule (e.g., a transgene) can encode a peptide, polypeptide, or protein that can be useful for the treatment or prevention of disease in a subject.

[0130] A heterologous nucleic acid molecule (i.e., transgene) can be a gene editing molecule used for modifying a genomic locus of interest (i.e., target) in a cell. A modification can include a disruption, deletion, repair, mutation, addition, alteration, or modification of a gene sequence at a target locus in a gene. Examples of gene-editing molecules include, but are not limited to, endonucleases such as zinc finger nucleases (ZFNs), transcription activatorlike effector nucleases (TALENs), meganucleases, restriction endonucleases, recombinases, and Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) / CRISPR- associated (Cas) proteins.

[0131] In certain embodiments, an AAV vector or expression cassette can be multi-cistronic. A multi-cistronic vector or expression cassette can simultaneously express two or more (e.g., 2, 3, 4, 5, 6, or more) separate proteins from the same mRNA. In such cases, the multiple heterologous nucleic acid molecules can be separated by an element that allows for separate translation for each gene.

[0132] In certain embodiments, a large heterologous nucleic acid molecule can be split into more than one construct, whereby multiple constructs can express two or more fragments for assembly into a protein of interest in vivo.

[0133] In certain embodiments, AAV vectors or expression cassettes do not include a reporter gene or a selection marker. In certain embodiments, the transgene-containing vectoror expression cassette does not comprise an antibiotic resistance gene. An AAV vector or expression cassette can include a stutter sequence as described above.

[0134] In certain embodiments, an AAV vector or expression cassette includes a Cap coding nucleic acid molecule and a Rep coding nucleic acid molecule.

[0135] In certain embodiments, one or more nucleic acid molecules of an AAV vector or expression cassette can be stably integrated into a genome of a cell line for AAV production, as a provirus. In certain embodiments, a heterologous nucleic acid molecule, such as a transgene, flanked by ITR sequences for packaging into an AAV capsid, a Cap coding nucleic acid molecule and a Rep coding nucleic acid molecule of an AAV vector or expression cassette can be stably integrated into a genome of a cell line for AAV production. In certain embodiments, a heterologous nucleic acid molecule, such as a transgene, flanked by ITR sequences for packaging into an AAV capsid is provided by an EBV vector that is maintained within the cell as an extrachromosomal element (e.g., as an EBV based nuclear episome).AAV PRODUCTION COMPOSITIONS

[0136] Materials and methods of the present disclosure can include compositions and systems for producing AAV. A composition or system for AAV production can include two or more vectors comprising: (i) one or more nucleic acid molecules encoding an Ad L4-33K protein; (ii) one or more nucleic acid molecules encoding Ad genes E2A, L4, E4, and VA RNA; (iii) one or more nucleic acid molecules encoding an AAV capsid (Cap) protein and one or more nucleic acid molecules encoding an AAV replication (Rep) protein; and (iv) one more nucleic acid molecules encoding a transgene flanked by ITR sequences. A vector can include one or more of the nucleic acid molecules in any combination that does not impair AAV production.

[0137] In certain embodiments, an AAV production composition or system can include a first vector or adenovirus comprising at least one nucleic acid molecule encoding an adenovirus L4-33K protein; and a second vector or adenovirus comprising Ad genes as described above. A nucleic acid molecule encoding an adenovirus L4-33K protein can be operatively linked to an EF1A promoter. In certain embodiments, Ad genes E2A, L4, E4, and VA RNA are serotype Ad2 or Ad5. In certain embodiments, a second vector or adenovirus further includes an Ad pIX gene. In certain embodiments, a second vector or adenovirus does not include an Ad E1A or E1 B gene. In certain embodiments, the first and second vectors or adenovirus can be included at a specific molar ratio. For example, the molar ratio of the first vector or adenovirus to second vector or adenovirus can be between 10:1 to 1 :10, between 5:1 and 1 :5, between 3:1 and 1 :3, between 2:1 and 1 :2; or between 1.5:1 and 1 :1.5. In certain embodiments, the molar ratio of the first and second vectors or adenovirus can be about 1 :1.

[0138] In certain embodiments, an AAV production composition or system can further include a third vector or adenovirus comprising a nucleic acid molecule encoding AAV replication (Rep) and AAV capsid (Cap) proteins. In certain embodiments, a third vector or adenovirus can encode Rep and Cap proteins of the same AAV serotype, or different serotypes. In certain embodiments, a third vector or adenovirus can include a nucleic acid molecule encoding a transgene flanked by ITR sequences. In some embodiments, a third vector or adenovirus can be an AAV transfer plasmid as described above. In certain embodiments, the molar ratio of first vector or adenovirus to third vector or adenovirus can be between 10:1 to 1 :10, between 5:1 and 1 :5, between 3:1 and 1 :3, between 2:1 and 1 :2; or between 1.5:1 and 1 :1.5. In certain embodiments, the molar ratio of first vector or adenovirus to third vector or adenovirus can be about 1 :1. In certain embodiments, an AAV production composition can include a first, second and third vector or first, second, and third adenovirus at a predetermined molar ratio within the ranges of between 10-1 :10-1 :10 to 1. In certain embodiments, a molar ratio of first, second, and third vectors or adenovirus can be about 1 :3: 1 or about 1 :1 :1.

[0139] The certain embodiments, an AAV production composition or system can include a fourth vector comprising a nucleic acid molecule encoding a transgene flanked by inverted terminal repeat (ITR) sequences. In some embodiments, a fourth vector or adenovirus can include least one heterologous nucleic acid molecule of interest (i.e., transgene) flanked by ITR sequences for packaging into an AAV capsid.

[0140] In certain embodiments, the molar ratio of second vector or adenovirus to fourth vector or adenovirus can be between 10: 1 to 1 : 10, between 5: 1 and 1 :5, between 3: 1 and 1 :3, between 2:1 and 1 :2; or between 1.5:1 and 1 :1.5. In certain embodiments, the molar ratio of second vector or adenovirus to fourth vector or adenovirus can be about 1 :1. In certain embodiments, an AAV production composition can include a first, second, and fourth vector or adenovirus at a predetermined molar ratio within the ranges of between 10-1 :10-1 :10 to 1. In certain embodiments, a molar ratio of first, second, and third vectors or adenovirus can be about 1 :3:1 or about 1 :1 :1.

[0141] In certain embodiments, the components of an AAV production composition or system can be separately packaged as a kit. In certain embodiments, a kit includes separately packaged first, second, third, and / or fourth vectors. In certain embodiments, a kit can include instructions for mixing or otherwise combining the vectors for the production of AAV.AAV PRODUCTION SYSTEMS

[0142] Materials and methods of the present disclosure can include systems for producing AAV. An AAV production system can include one or more nucleic acid molecules described above that are sufficient, when introduced into a suitable host cell (either by incorporation inthe genome or by transient transfection), to support production of AAV and a nucleic acid molecule encoding an Adenovirus (Ad) L4-33K protein (i.e., an L4-33K-encoding nucleic acid molecule ). In certain embodiments, an AAV production system can include nucleic acid molecules encoding AAV Rep and Cap proteins, nucleic acid molecules encoding helper genes, and an AAV construct, a transgene expression cassette, or AAV transfer plasmid.

[0143] An AAV production system can include (a) a cell; (b) a first vector comprising at least one nucleic acid molecule encoding an Ad L4-33K protein; (c) a second vector comprising one or more nucleic acid molecules encoding Ad genes E2A, L4, E4, and VA RNA; and (d) a third vector comprising one or more nucleic acid molecules encoding AAV Rep and AAV Cap proteins and a transgene flanked by inverted terminal repeat (ITR) sequences; or a third vector comprising one or more nucleic acid molecules encoding AAV Rep and AAV Cap proteins and a fourth vector comprising a transgene flanked by ITR sequences. A cell can be an Sf9 cell, CHO, cell, Vero cell, HeLa cell, MDCK cell, BHK cell, A549 cell, amniocyte (e.g., an amniocyte production cell such as CAP® or CAP-T® (CEVEC)), or HEK293 cell. In certain embodiments, a cell can express Ad E1A and / or Ad E1 B genes (e.g., a HEK293 cell). In certain embodiments, the third vector can further include a selectable marker. Exemplary plasmids containing one or more nucleic acid molecules encoding AAV Rep and AAV Cap proteins, a transgene flanked by ITR sequences, and a selectable marker is described in Martin et al., Human Gene Therapy Methods, 24: 253-259 (2013), and Luo et al., Human Gene Therapy Methods, 28(3): 124-138, which are incorporated by reference herein.

[0144] In certain embodiments, an AAV production system can be used to produce AAV via a multi-plasmid transfection method, such as a triple transfection method. An exemplary triple transfection method is described in Adsero et al., Human Gene Therapy, 35(1-2):59-69 (2024). An AAV production system can include an L4-33K-encoding nucleic acid molecule, nucleic acid molecules encoding AAV Rep and Cap proteins, nucleic acid molecules encoding helper genes, and an AAV construct, a transgene expression cassette, or AAV transfer plasmid comprising the same plasmid backbone. In certain embodiments, at least one of the three plasmid backbones comprising an L4-33K-encoding nucleic acid molecule, nucleic acid molecules encoding AAV Rep and Cap proteins, nucleic acid molecules encoding helper genes, and an AAV construct, a transgene expression cassette, or AAV transfer plasmid is different. In certain embodiments, all plasmid backbones are different. In certain embodiments, an AAV production system includes plasmid backbones based on, for example and without limitation, pUC19, pBR322, pUC57, pJ24l, or pJ247. In certain embodiments, at least one of the plasmids comprise plasmid backbones can be based on pUC57.

[0145] In certain embodiments, one plasmid includes an L4-33K-encoding nucleic acid molecule, a second plasmid serves as an AAV Rep-Cap construct, and a third plasmid can include at least one heterologous nucleic acid molecule of interest (i.e., transgene) flanked byITR sequences for packaging into an AAV capsid. In certain embodiments, a third plasmid including at least one heterologous nucleic acid molecule of interest flanked by ITR sequences can be referred to as a “Triple-play plasmid” (TTP). In certain embodiments, the molar ratio of a TTP to a plasmid including a nucleic acid molecule encoding helper genes (e.g., a plasmid that can be referred to as a “pAdhelper”) can be between 10:1 to 1 :10, between 5:1 and 1 :5, between 3:1 and 1 :3, between 2:1 and 1 :2; or between 1.5:1 and 1 :1.5. In certain embodiments, the molar ratio of the TTP and pAdhelper can be about 1 :1. In certain embodiments, a pAdHelper can include and / or be supplemented with an L4-33K-encoding nucleic acid molecule.

[0146] In certain embodiments, one plasmid includes an L4-33K-encoding nucleic acid molecule, a second plasmid serves as an AAV Rep-Cap vector, a third plasmid can include an AAV expression cassette comprising least one transgene flanked by ITR sequences, and a fourth plasmid serves as a helper vector comprising one or more nucleic acid molecules encoding Ad genes E2A, L4, E4, and VA RNA.

[0147] In certain embodiments, an AAV production system includes nucleic acid molecules encoding AAV Rep and Cap proteins and nucleic acid molecules encoding helper genes supplied by a single Ad vector.

[0148] One or more of the vectors or expression cassettes described above can contain a selection marker. An example of a selection marker includes, but is not limited, to positive selection markers such as drug resistance genes including, but not limited to, G418 (with neor), puromycin (with puror), hygromycin B (with hygr), blasticidin S (with bsrr), mycophenolic acid and 6-thio(guanine) (with gpt) and gancyclovir or 1 (2'-deoxy-2'-fluoro-beta-D- arabinofuranosyl)-5-iodouracil (Fl AU) (with HSV-tk), gentamycin, and / or kanamycin (with kanr). In a further embodiment, a selection marker can be kanamycin.

[0149] One or more of the plasmids described above can contain one or more reporter genes. A reporter gene can be inserted within a vector based on suitability for a specific organism, under the control of a promoter. Examples of reporter genes include, without limitation, b-galactosidase (LacZ), firefly luciferase, Renilla luciferase, Gaussia luciferase, chloramphenicol acetyltransferase (CAT), secreted embryonic alkaline phosphatase (SEAP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), enhanced GFP (eGFP), yellow fluorescent protein (YFP), enhanced YFP (eYFP), blue fluorescent protein (BFP), enhanced BFP (eBFP), and red fluorescent protein from the Discosoma coral (DsRed). A reporter construct can include a combination of reporter genes.

[0150] One or more of the vectors described above can include a DNA titer tag. For example, DNA titer tag can be included in an AAV transfer plasmid, or in each of the different plasmids.

[0151] An AAV production system can include a cell for producing AAV. A cell for producing AAV can be any cell that is suitable for transient or stable transfection with the nucleic acidmolecules or vectors described above, and optionally, also susceptible to infection by a helper virus. A cell for producing AAV can be a cell that has been transiently or stably transfected with one or more of the nucleic acid molecules described above.

[0152] In certain embodiments, a cell for AAV production can be capable of producing one or more AAV serotypes, or chimeras or hybrids thereof, e.g., AAV1 , AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, chimeric vectors, and variants thereof. In certain embodiments, a cell for AAV production can be a Sf9 cell, CHO cell, Vero cell, HeLa cell, MDCK cell, BHK cell, A549 cell, or HEK293 line. In certain embodiments, cells for producing AAV can be suspension cells such as insect cell lines Sf9 or H5 cells, HeLa based cell lines, BHK21 cells lines, and suspension adapted HEK 293 cells, or adherent cells, such as HEK 293 cells. In certain embodiments, an AAV production system includes HEK 293 cells.

[0153] In certain embodiments, and AAV production system can include a cell line having a genome stably integrated with one or more nucleic acid molecules encoding AAV Rep and AAV Cap proteins, an Ad L4-33K protein, a transgene flanked by inverted terminal repeat (ITR) sequences; and Ad genes E2A, L4, E4, and VA RNA. In certain embodiments, the Ad genes are serotype Ad2 or Ad5. A cell line can be an Sf9 cell line, Vero cell line, HeLa cell line, MDCK cell line, BHK cell line, A549 cell line, or HEK293 cell line. In certain embodiments, a cell line can be a HeLa S3 or HEK293 cell line. An AAV production system can produce serotype AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12 or AAV13, a variant thereof, or a pseudo-serotype AAV.METHODS OF PRODUCING AAV

[0154] The present disclosure provides methods of producing AAV with supplementation of an Ad L4-33K protein, using a nucleic acid molecule as described above. In certain embodiments, a method of producing AAV can include (a) integrating into a cell genome one or more nucleic acid molecules encoding AAV Rep and AAV Cap proteins, a transgene flanked by inverted terminal repeat (ITR) sequences; and an Ad L4-33K protein; (b) integrating into the cell genome adenovirus genes E2A, L4, E4, and VA RNA or transfecting the cell line with an adenovirus; (c) expanding the cell and producing the AAV within the cell; and (d) isolating the AAV. A method of the present disclosure can produce a higher AAV titer (vg / ml) than a corresponding method that does not include integration of the one or more nucleic acid molecules encoding the adenovirus L4-33K protein into its genome.

[0155] A method of producing AAV can include transfecting a cell with two or more vectors comprising: (i) one or more nucleic acid molecules encoding an Ad L4-33K protein; (ii) one or more nucleic acid molecules encoding adenovirus genes E2A, L4, E4, and VA RNA; (iii) one or more nucleic acid molecules encoding an AAV capsid (Cap) protein and one or more nucleicacid molecules encoding an AAV replication (Rep) protein; and (iv) one more nucleic acid molecules encoding a transgene flanked by inverted terminal repeat (ITR) sequences; culturing a transfected cell under conditions suitable for producing AAV; and isolating the AAV.

[0156] In certain embodiments, integrating includes contacting a cell with an AAV production composition as described above (e.g., a multi-plasmid transfection technique). In certain embodiments, an AAV production composition can be provided to a cell culture under conditions suitable for producing an AAV, i.e. , a viral particle comprising recombinant singlestranded DNA containing a heterologous nucleic acid molecule, such as a transgene within an AAV capsid in the cell. In certain embodiments, the cell culture is a suspension culture.

[0157] In certain embodiments, a method of producing AAV can include a step of collecting AAV from a cell culture. In certain embodiments, an AAV can be collected by lysing the cells, e.g., after removing the cells from the culture medium, e.g., by pelleting the cells. In another embodiment, the virus vector can be collected from the medium in which the cells are cultured, e.g., to isolate vectors that are secreted from the cells. Some or all of the medium can be removed from the culture one time or more than one time, e.g., at regular intervals during the culturing step for collection of AAV (such as every 12, 18, 24, or 36 hours, or longer extended time that is compatible with cell viability and vector production), e.g., beginning about 48 hours post-transfection. After removal of medium, fresh medium, with or without additional nutrient supplements, can be added to a culture. In one embodiment, the cells can be cultured in a perfusion system such that medium constantly flows over the cells and is collected for isolation of secreted AAV. Collection of AAV can continue for as long as the transfected cells remain viable, e.g., 48, 72, 96, or 120 hours or longer post-transfection. In certain embodiments, the collection of secreted AAV is carried out with serotypes of AAV (such as AAV8 and AAV9), which do not bind or only loosely bind to a cell. In other embodiments, the collection of secreted AAV is carried out with heparin binding serotypes of AAV (e.g., AAV2) that have been modified so as to not bind to the cells in which they are produced.

[0158] In certain embodiments, a method of producing AAV includes culturing a contacted cell in any volume of culture medium, from 10 ml (e.g., in shaker flasks) to 10 L, 50 L, 100 L, or more (e.g., in bioreactors).

[0159] In certain embodiments, an AAV production method with supplementation of an Ad L4-33K protein provides at least about 1 *104vector genome-containing particles per cell prior to purification, e.g., at least about 2*104, 3x104, 4*104, 5x104, 6x104, 7x104, 8x104, 9x104, or 1 xl05or more vector genome-containing particles per cell prior to purification. In certain embodiments, the number of vector genome-containing particles provided is enhanced as compared with a method that does not include supplementation of an Ad L4-33K protein. For example, expression of a L4-33K protein and Ad helper genes E2A, L4, E4, and VA RNA can enhance AAV production in vector genomes per ml (vg / mL) as compared to a cell that doesnot express a L4-33K protein and Ad helper genes E2A, L4, E4, and VA RNA. In certain embodiments, supplementing cell expressing Ad helper genes E2A, L4, E4, and VA RNA by expressing a L4-33K protein can provide at least about 1 x1012purified vector genomecontaining particles per liter of cell culture, e.g., at least about 5x1012, 1 X 1013, 5x1013, or 1 xl014or more purified vector genome-containing particles per liter of cell culture. In certain embodiments, an enhancement in AAV production can be greater than 1.25-fold, 1.5-fold, 2- fold, 3-fold, 5-fold or 10-fold over the AAV titer of an AAV production method without Ad L4- 33K protein supplementation. In certain embodiments, AAV production can be enhanced by 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0- fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4.0-fold, 4.1-fold, 4.2-fold, 4.3-fold, 4.4-fold, 4.5-fold, 4.6-fold, 4.7-fold, 4.8-fold, 4.9-fold, 5.0-fold, 5.1- fold, 5.2-fold, 5.3-fold, 5.4-fold, 5.5-fold, 5.6-fold, 5.7-fold, 5.8-fold, 5.9-fold, 6.0-fold, or greater as compared to the AAV titer of an AAV production method without Ad L4-33K protein supplementation.

[0160] In certain embodiments, a method of producing AAV can include removing helper virus (e.g., Ad vectors). In certain embodiments, AAV can be separated by size or affinity.

[0161] In certain embodiments, a method of producing AAV can include evaluation of the AAV. For example, evaluation can include determining the number of replication-competent AAV or determining the number of AAV that result in the production of a functional transgene product. Functional assays can include those described in Examples herein, or for example, in Xiao et al. (1997) Exp. Neurobiol., 144: 113-124; or in Fisher et al. (1996) J. Virol, 70:520- 532 (LFU assay) and McLaughlin et al. (1988) J. Virol., 62: 1963-1973 (describing the replication center assay).AAV COMPOSITIONS AND METHODS OF USE

[0162] AAV produced according to one or more embodiments of the present disclosure can be used for the delivery of nucleic acids to cells in vitro, ex vivo, and in vivo. AAV can be advantageously employed to deliver or transfer nucleic acids to animal cells, including mammalian cells.

[0163] In certain embodiments, a heterologous nucleic acid molecule can encode any polypeptide or RNA for production in a cell in vitro, ex vivo, or in vivo. For example, nucleic acids of interest include nucleic acids, such as genes, encoding polypeptides or RNAs. In certain embodiments, an AAV can be used to deliver a reporter, therapeutic (e.g., for medical or veterinary uses), immunogenic (e.g., for vaccines), or diagnostic polypeptide or RNA. In certain embodiments, an AAV can be used to introduce a heterologous nucleic acid sequence into cultured cells and the expressed gene product isolated therefrom.

[0164] An AAV produced according to the present disclosure can be used for delivering heterologous nucleic acids into a broad range of cells, including dividing and non-dividing cells. An AAV can be employed to deliver a nucleic acid of interest to a cell in vitro, e.g., to produce a polypeptide in vitro or for ex vivo gene therapy. An AAV can be useful in a method of delivering a nucleic acid to a subject in need thereof, e.g., to express an immunogenic or therapeutic polypeptide or a functional RNA in a subject. A subject can be in need of a beneficial effect provided by expression of an AAV delivered immunogenic or therapeutic polypeptide or a functional RNA. In certain embodiments, AAV can be used to produce a polypeptide of interest or functional RNA in cultured cells or in a subject to observe the effects of a polypeptide or functional RNA on a subject, for example, in connection with screening methods.

[0165] The present disclosure is also described and demonstrated by way of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only.EXAMPLES

[0166] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions featured in the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.

[0167] Abstract: Elucidating the function of Adenovirus late gene products and impact on AAV production.

[0168] Recombinant adeno-associated viral vectors (rAAV) are being developed for a wide variety of gene therapy applications, but efficient production at large scale remains challenging. The producer cell line (PCL) platform is an economical and scalable approach for rAAV manufacturing. AAV Rep / Cap genes and gene of interest (GOI) elements are stably integrated in the producer cell line, and rAAV production is induced by infection with a wild-type, replication-competent Adenovirus (wtAd5). The adenoviral helper function in AAV production has been studied extensively, however, the interplay of the AAV and wtAd5 genomes is not yet fully understood. To better understand the helper function provided by wtAd5 in the PCL platform, the Examples below utilized a PCL infected with a variant Adenovirus serotype 5 virus (Ad5ts149), which harbors a temperature-sensitive mutation in the Ad5 polymerase gene E2B. Suppression of Ad5ts149 replication and packaging at the restrictive temperature (39°C) resulted in a 3-to-50-fold reduction in rAAV production compared to that of wtAd5. However, replication of the AAV genome was comparable or higher in Ad5ts149-infected cells.

[0169] AAV Rep / Cap protein expression was analyzed to assess the underlying mechanism. An altered Rep expression pattern was detected, accompanied by reduced expression of Cap protein. Orthogonal methods were employed to further understand transcription of AAV and adenoviral genes in the context of an Ad5ts149 infection at 39°C. Analysis of Ad5ts149 gene expression via RT-qPCR showed that expression of the minimal essential Ad helper genes were similar to wtAd5 while the expression of the Ad late gene products was decreased. Hypotheses were tested via siRNA knockdown in conjunction with wtAd5 infection to gain insight on the role of Ad5 late gene products. The outcomes reveal a novel role of an Ad5 late gene product in the regulation of AAV rep / cap gene expression and efficient production of rAAV in an exemplary AAV producer cell line platform.

[0170] Example 1 : rAAV production when suppressing Ad replication and production with adts149 at 39°C in PCL.

[0171] Producer cell line (PCL) cells were seeded in spin tubes and infected with either wtAd5 or adts149 at specified multiplicities of infection (MOI). Temperature-sensitive AdV mutant adts149 (also referred to as “ad5ts149” or “Ad5ts149” throughout the present disclosure) is defective for late gene expression and has a low replication rate at 39°C, which decreases adenoviral load in the preparations. Whole cell culture were harvested after 48 hours post-infection and cell lysates were quantified by qPCR using target-specific primer / probe sets for AAV or Ad titers. The results are shown in Figure 1. AAV production was significantly reduced when suppressing Ad replication and production with adts149 at 39°C (Figure 1 A). AAV replication was also decreased but to a less extent compared to that of AAV production(Figure 1B). Figure 1C and 1D show the Ad replication and production was decreased when using adts149 at 39°C. DRG: DNase-resistant genome copies; TVG: total genome copies. 37°C: Adts149 semi-permissive temperature; 39°C, Adts149 non-permissive temperature.

[0172] Example 2: Suppression of Ad replication and production with Adts149 at 39°C alters Rep / Cap transcription, splicing and protein expression.

[0173] Infected PCL samples were collected as described in Example 1 for rep / cap expression analysis. Total cell lysates were subjected to Western analysis using anti-Rep, anti-Cap, and anti-beta-tubulin antibodies as shown in Figure 2A. Altered Rep expression pattern and significantly reduced Cap expression was associated with adts149 at 39°C. Spliced Rep68 and Rep40 as well as VP3 expression was significantly decreased. Figure 2B shows the amplified Rep / Cap copy numbers quantified by dPCR using extrachromosomal DNA extracted from Ad-infected cells. The Rep / Cap amplification was not substantially different between adts149 and wtAd at restricted temperatures. Total RNA isolated from Ad- infected cells were subjected to Northern blot analysis with a Cap-specific probe. Rep / Cap transcripts were separated and visualized in Figure 2C (left). A pattern shift toward less splicedRep / Cap transcripts was observed from adts149 at restricted temperature (39°C), consistent with reduced spliced form Rep68 / 40 and VP3 expression. Rep / Cap spliced transcripts were quantified via RT-qPCR with target-specific probes and normalized to GAPDH. Rep / Cap splicing percentages were calculated by normalizing spliced transcripts numbers to total Rep / Cap transcripts using primers / probes illustrated in Figure 2C (right). The relative splicing percentage and fold change of Rep / Cap mRNA are shown in Figure 2D and 2E. Suppression of Ad replication and production decreased the overall splicing ratio and reduced the Rep / Cap mRNA expression compared to non-restricted temperature (37°C).TABLE 1. Primer / probe sequences.

[0174] Example 3: Ad gene expression profiling identifies reduced Ad L4 gene expression at 39°C with adts149.

[0175] Infected PCL samples were collected as described in Example 1 for Ad gene expression profiling. Expression of minimum essential Ad genes, and Ad late gene including Hexon and L4 products were quantified using RT-qPCR and normalized to GAPDH. The fold change in expression of Ad E1A (Figure 3A), E1B55K (Figure 3J), E1B19K (Figure 3K), E2A (Figure 3C), E4orf6 / 7 (Figure 3L), VA RNA (Figure 3M), E2B (Figure 3N), HEXON (Figure 3E), L4-100 / 33 / 22K (Figure 3G), L4-100K (Figure 3H), L4-100 / 22K (Figure 3I) was plotted relative to that of wtAd- infected cells incubated at 37°C.

[0176] Total cell lysates were subjected to Western blot analysis using anti-E1A(Figure 3B), anti-Ad5 E2A (Figure 3D), anti-Ad5 capsid (Figure 3F). Expression of minimum essential helper genes was comparable for adts149 at 39°C to that of wtAd. However, Ad late gene expression, for example Ad L4, was reduced with adts149 at 39°C.

[0177] Example 4: Knockdown of L4 gene expression reduces rep / cap transcription, splicing and expression.

[0178] Ad L4 gene knockdown studies were performed in producer cell line via transient transfection of DsiRNA using nucleofection. Target-specific DsiRNA were ordered from IDT according to Su et al 20243. PCLs were infected with wtAd immediately upon nucleofection and harvested at 72 hours post infection for downstream analysis. Total RNA were extracted and subjected for RT-qPCR analysis with target-specific primer / probe sets using GAPDH as the internal control. The fold change of target mRNAs upon DsiRNA knockdown were compared to a non-targeting siRNA negative control. Figure 4A shows the illustration of the dsiRNA targets. The illustration was adapted from Biasiotto et al 20154and Su et al 20243and made with BioRender™. Figure 4B and 4C demonstrates the knock down efficiency for L4 targets were at least 90%. As shown in Figure 4D, knockdown of 22 / 33K reduced rep / cap transcription by 100 fold. Figure 4E shows knockdown of L4 genes decreased rep / cap splicing from 75% to around 50%. Rep / Cap protein expression were analyzed via Western blotting as described in Example 2. Rep / Cap expression was significantly reduced when knocking down 22 / 33K. AAV Titer was determined for each sample and normalized to the non-targeting siRNA negative control as shown in Figure 4G. The AAV production was significantly suppressed when suppressing L4 22 / 33K expression.

[0179] Example 5: Supplementation of Ad L4-33K improves Rep / Cap splicing, expression and AAV production in the context of Adts149 infection at 39°C.

[0180] AAV producer cells were transfected with L4 constructs expressing individual genes L4-100K, 22K, 33K or pVIII driven by EF1A promoter (Figure 5A) using nucleofector 4D. Transfected cells were infected at 24 hrs post transfection with adts149 at 15IU / cell and incubated at 39°C. Cells were harvested for downstream analysis at 48 hrs after infection. Total RNA were extracted and subjected for RT-qPCR analysis with target-specific primer / probe sets using GAPDH as the loading control. The fold change of mRNA were compared to the pUC57 stuffer control. AAV Rep / Cap splicing were assessed by normalizing spliced transcripts copy number to total transcripts number from RT-qPCR. Figure 5B shows the splicing percentage value for each condition and additional L4-33K supplementation enhanced the rep / cap splicing. Figure 5C shows relative Rep / Cap mRNA expression comparing different L4 gene supplementation to that of pUC57 control. Rep / Cap protein expression were analyzed via Western blotting as described in Example 2. As shown in Figure 5D, upon 33K supplementation, Cap expression was significantly enhanced associated with more spliced Rep68 and Rep40 expression. AAV Titer was determined for each sample and normalized to the pUC57 control as shown in Figure 5E. L4-33K overexpression increased rAAV productivity by 3-fold.

[0181] Example 6: Supplementation of additional L4-33K increases Rep / Cap splicing and rAAV production in HEK293 Transient Transfection Platform.

[0182] pAAV with Rep / Cap / transgene, pAdhelper and EF1a promoter driven L4 construct or pUC57 were co-transfected into HEK293 at molar ratio of 1 :1 :1 as shown in Figure 6A. Cultures were harvested 72 hrs post transfection for downstream analysis. Total RNA were extracted and subjected for RT-qPCR analysis with target-specific primer / probe sets using GAPDH as the internal control. The fold change of mRNA was compared to the pUC57 stuffer control. Relative splicing percentage were calculated by dividing the number of spliced transcripts to total Rep / Cap transcripts using primers / probes specific to spliced and total transcripts respectively. Figure 6B shows the splicing percentage value for each condition and additional L4-33K supplementation enhanced the rep / cap splicing. Figure 6C shows the fold change value for Rep / Cap mRNA transcripts. AAV Titer was determined for each sample and normalized to the pUC57 control as shown in Figure 6D. Additional L4-33K overexpression increased rAAV productivity by 1.5-fold in for the tested capsid serotype. Enhanced rAAV production was also seen when 33K supplementation was tested with three additional capsids / transgenes as shown in Figure 6E.MATERIALS AND METHODS

[0183] Cells and cell culture

[0184] AAV producer cell line was generated and cultured as previously reported1. Briefly, parental host cells were cultured in Excell-HeLa media (Sigma-Aldrich) supplemented with 6mM Glutamine in suspension shake flask at 3 7°C with 5% CO2, 80% humidity at 100rpm. A plasmid containing AAV Rep / Cap expression cassettes, ITR-transgene and Puromycin selection maker was stably transfected into parental host cells. Clonal producer cell lines were screened for best AAV productivity and scaled up in the culture medium. Producer cell cultures were maintained by passaging at every 3-to-4-day intervals and seeded for intended experiments generally from a 3-day scale up culture.

[0185] HEK293 cells were cultured following manufacture protocol. Briefly, HEK293 cells were cultured in LVMax production medium (Thermo Fisher) in suspension shake flask at 37°C with 8% CO2, 80% humidity at 125rpm. Cultures were passaged every 3-to-4 days and seeded at target seeding density for transient transfection generally from a 3-day culture.

[0186] Viruses and virus infection

[0187] Wild type Adenovirus type 5 (wtAd5, ATCC-VR5) and Adts149, an Ad5 mutant harboring temperature sensitive mutation within E2B were used as helper viruses to infect producer cell lines. For routine PCL infection, cells were seeded at targeted density in proprietary production media and infected with wtAd5 at multiplicity of 100 DNase-resistant genome particles per cell2. For Adts149 infection, three MOIs (3.74, 15 and 100) were used to infect producer cells at either semi-permissive 37°C or non-permissive 39°C. Infections were performed in suspension spin tubes or shake flasks and cultured at 37°C, 10% CO2.

[0188] Plasmid, DsiRNA and transfection

[0189] For Ad L4 gene overexpression, constructs expressing each of Ad L4 genes from EF1A promoter including EF1A-33K, EF1A-100K, EF1A-22K, EF1 A-pVI II were ordered and synthesized from Genscript using pUC57 backbone. The coding sequences for 33K and 22K genes are SEQ ID No: 2 and 5, respectively.

[0190] For Ad L4 gene knockdown, DsiRNA targeting different L4 regions including 100K, 22K / 33K3were ordered from IDT along with negative control DsiRNA (IDT, cat# 51011403).

[0191] For PCL transfection, Lonza 4D nucleofector and SE Cell Line 4D-Nucleofector X L (Lonza, cat# V4XC1024) was used for DsiRNA and plasmid DNA transfection. Transfections were performed following Manufacture’s protocols. For HEK293 transfection, Polyplus FectoVIR was used to transfect serum free suspension HEK293 with DNA amount of 0.5 pg pDNA per 1 E6 viable cells. Transfection complex was generated using plasmid DNA to FetoVIR ratio of 1 at 5% complexing volume. Transfection complex was then added into HEK293 cells within 25min of complexing incubation and cells were returned to shaker incubator for continued culturing.

[0192] AAV production and titration

[0193] AAV production from PCLs were initiated by infecting producer cells with a helper virus, such as wild type Ad5 (wtAd5) as described above. AAV replication and production was analyzed with 3 days post infection whole culture cell lysates.

[0194] AAV production from HEK293 transient transfection was performed as described below. For HEK293 AAV production transfection, three plasmid DNAs including pAdhelper (including E2A, E4, L4 and VA RNAs), pAAV-Rep / Cap / Transgene, and pEF1A-L4 constructs or pUC57 stuffer control were mixed at 1 :1 :1 ratio and transfected as described above. Transfected cultures were collected at 3dpt for AAV replication and production analysis.

[0195] AAV replication and production were analyzed as previously described1. Briefly, whole production cultures were collected and lysed with Tween®-20 (polysorbate) (0.5%) followed by treatment with Benzonase (10U / ml) for AAV production titer analysis. Benzonase treated cell lysates were further digested with DNase (Promega; 60 U / ml final concentration) for 10 min at 37°C temperature. The DNase reaction was terminated via addition of 1 mg / ml Proteinase K (Invitrogen; 0.5 mg / ml final concentration) and incubation at 65°C for 10 min followed by heat inactivation at 95°C for 20 min. Heat-inactivated post Proteinase K samples were then diluted and analyzed with Quantstudio 7flex using BGH polyA following standard qPCR procedures. AAV genome copy numbers were calculated via comparison to a standard curve generated with a plasmid (pAF196) that contained all PCR target sequences (e.g., rep, cap, BGH polyA, Ad E2A). For analysis of total DNA copies, the same procedure was followed without Benzonase or DNase treatment.

[0196] Gene expression analysis

[0197] Rep / Cap gene amplification analysis:

[0198] Rep / Cap gene stably integrated in producer cell lines undergoes amplification after Ad5 helper virus infection. The amplified copies were suggested to exist as extrachromosomal DNA (ecDNA). To assess Rep / Cap amplification, ecDNA were extracted from infected producer cell pellets using Purelink Hipure DNA extraction mini kit (Life technology cat# K210003). To quantify rep / cap amplification, duplex dPCR using Rep / Cap specific primer / probes were performed using ecDNA as the template. The dPCR was carried out with QIAcuity Probe PCR Kit (Qiagen cat# 250102) according to manufacturer’s protocol on a QIAcuity 8. Rep / Cap gene copies were normalized to per cell number for comparison.

[0199] Gene expression transcription analysis:

[0200] To analyze AAV Rep / Cap mRNA transcription, total RNA was isolated from infected producer cell line or transfected HEK293 cells using RNeasy plus kit (Qiagen cat# 74134) according to manufacturer’s instructions and used for further analysis.

[0201] For AAV rep / cap total transcripts analysis, Northern blot was performed with 3-4 ug total RNA using the NorthernMax-Gly kit (Invitrogen Cat# AM1946). A digoxigenin (DIG)- labeled cap specific probe was prepared using the PCR DIG probe Synthesis kit (Roche, cat# 11636090910) according to the manufacturer’s instructions. The blot was UV-crosslinked and pre-hybridized in EasyHyb solution (Roche, Cat# 11603558001) for 30 min before hybridization with denatured probe at 56°C overnight. The blot was then washed and developed using the DIG Wash and Block buffer set (Roche, cat# 11585762001) and DIG luminescent Detection kit (Roche, Cat# 11363514910), according to the manufacturer’s protocol.

[0202] For quantification of Rep / Cap spliced transcripts, total RNA was reverse transcribed to cDNA using SuperScript™ IV VILO™ Master Mix with ezDNase™ Enzyme (Invitrogen cat# 11766050) followed by quantification via dPCR with QIAcuity Probe PCR Kit (Qiagen cat# 250102) on a QIAcuity 8 (Qiagen). Serial dilution of cDNA reaction was used as the template for dPCR using primers / probes targeting total Rep, Cap transcripts or spliced specific transcripts. GAPDH was used as the internal loading control. AAV rep / cap splicing efficiency was assessed using dPCR values obtained from primers / probe targeting splicing specific transcripts divided by values from primers / probe targeting total Rep / Cap transcripts.

[0203] Ad related mRNA was analyzed with QIAcuity EG PCR kit (Qiagen cat# 250112) with target-specific primers.

[0204] Western blot protein analysis:

[0205] Cell pellets were lysed in RIPA lysis and Extraction buffer (Life technology Cat# 89900) supplemented with Benzonase (10U, EMD cat# 1.01697.0001), MgCh (2mM) and Halt protease inhibitor cocktail (Thermo cat# 78425). Total cell lysates were subjected toelectrophoresis in a 4-12% NuPAGE Bis-Tris gel in NuPAGE MOPS running buffer (Life Technology Cat# NP0001) and then transferred to a nitrocellulose membrane (Nupage cat# IB23001)) using the iblot2 dry blotting system. The primary antibodies used were anti-AAV2 Rep (American research lab cat# 03-61071, 1:200), anti-AAV2 Cap (American research products, clone B1 cat# 690058, 1 :1000), anti-beta-tubulin Dylight 680 (Invitrogen Cat# NA516308D680), anti-Ad DBP (CUSABIO, cat# CSB-PA365892ZA01HIL), Anti-Ad-E1A (Santa Cruz cat# sc-25), and anti-Ad5 capsid (Abeam cat# AB6982). The secondary antibodies were Goat anti mouse H+L HRP (Biorad, cat# 1706516, 1:3000), Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP (Invitrogen, cat# 32460) and Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, DyLight™ 800 (Invitrogen cat# SA510036). The ECL (Biorad Cat# 1705060) was used for detection.REFERENCES (Examples 1-6)1. Martin, J. et al. Generation and Characterization of Adeno-Associated Virus Producer Cell Lines for Research and Preclinical Vector Production. Hum. Gene Ther. Methods 24, 253- 269 (2013).2. Luo, Y. et al. AAVS1 -Targeted Plasmid Integration in AAV Producer Cell Lines. Hum. Gene Ther. Methods 28, 124-138 (2017).3. Su, W., Seymour, L. W. & Cawood, R. AAV production in stable packaging cells requires expression of adenovirus 22 / 33K protein to allow episomal amplification of integrated rep / cap genes. Sci. Rep. 13, 21670 (2023).4. Biasiotto, Roberta & Akusjarvi, Goran. Regulation of Human Adenovirus Alternative RNA Splicing by the Adenoviral L4-33K and L4-22K Proteins. International Journal of Molecular Sciences. 16, 2893-2912 (2015).Example 7: Characterization of the function of Adenovirus L4 gene products and their impacts on AAV vector production.

[0206] The following studies further validate the results above as demonstrating that L4- 33K gene products impact AAV production in both the producer cell line and transient transfection platforms and that optimizing Ad L4-22K / 33K expression to facilitate efficient expression and splicing of AAV rep / cap transcripts represents a unique opportunity to optimize AAV vector production.

[0207] Materials and methods

[0208] Cells and cell culture

[0209] The AAV producer cell lines were generated and cultured as previously described (Martin, et al., 2013). Parental host cells were cultured in EX-CELL™ media (Sigma-Aldrich, St. Louis, MO, cat# 14591 C) supplemented with 6 mM Glutamine (Gibco, Billings, MT, cat# 25030081) in suspension shake flask at 100 rpm, 37°C with 5% CO2 and 80% humidity. Aplasmid containing the AAV rep / cap genes, the vector genome and a puromycin resistance gene was stably transfected into parental host cells. Clonal producer cell lines were screened for AAV productivity and scaled up in the culture medium. Producer cell cultures were maintained by passaging at 3-to-4-day intervals and seeded for intended experiments generally from a 3-day culture.

[0210] HEK293 cells were cultured following the manufacturer’s protocol. HEK293 cells were cultured in LVMAX™ production medium (Thermo Fisher, Waltham, MA, cat# A3583402) in suspension shake flask at 37°C with 8% CO2, 80% humidity at 120 rpm. Cultures were passaged every 3-to-4 days and seeded at target seeding density for transient transfection generally from a 3-day culture.

[0211] Viruses and virus infection

[0212] Wild type human Adenovirus type 5 (wtAd5, ATCC-VR5) and Ad5ts149, an Ad5 mutant harboring temperature sensitive mutation within E2B, were used as helper viruses to infect producer cell lines (Ensinger, et al., 1972; Stillman, et al., 1982). wtAd5 was produced and titrated according to standard procedures as reported previously (Martin, et al., 2013; Graham, et al., 1991). Ad5ts149 was grown at the permissive temperature (33°C) and purified as previously described (Vincent, et al., 1997). For wtAd5 infection, PCLs were seeded at the target density in proprietary production media and infected with wtAd5 at the indicated multiplicity of infection (MOI) (Luo, et al., 2017). For Ad5ts149 infection, three MOIs (4, 15 and 100) were used to infect producer cells at either the semi-permissive 37°C or non-permissive 39°C temperature. Infections were performed in suspension spin tubes or shake flasks and cultured at 37°C, 10% CO2 and 80% humidity.

[0213] Plasmid, DsiRNA and transfection

[0214] For adenoviral L4 gene overexpression, constructs of each of the human Adenovirus type 5 (AC-000008) L4 genes, including L4-100K (nucleotides nt 24061-26484), L4-22K (nt 26195-26785), L4-33K (nt 26195-26510 / 26713-27086), L4-pVIII (nt 27174-27857), expressed from the EF1a promoter was obtained from Genscript (Piscataway, NJ).

[0215] For L4 gene knockdown, DsiRNAs targeting different L4 regions including 100K, 22K / 33K as described in Su et al. (2023) were ordered from IDT (San Jose, CA) along with negative control DsiRNA (IDT, San Jose, CA, cat# 51011403).

[0216] For PCL transfection, Lonza 4D nucleofector and SE Cell Line 4D-Nucleofector X kit L (Lonza, Basel, Switzerland, cat# V4XC1024) were used for DsiRNA and plasmid DNA transfection. Transfections were performed following manufacturer’s protocols. For HEK293 transfection, Polyplus FectoVIR-AAV (lllkirch, France) was used to transfect serum free suspension HEK293 with 0.5 pg plasmid DNA per 1 E6 viable cells. The transfection complex was generated using a plasmid DNA to FectoVIR ratio of 1 at 5% complexing volume. The transfection complex was then added into HEK293 cells within 25 min of complexingincubation and cells were subsequently returned to the shaker incubator for continued culturing.

[0217] AA V production and titration

[0218] AAV production from PCLs were initiated by infecting producer cells with a helper virus as described above. AAV replication and production was analyzed 2-3 days post infection in whole culture cell lysates as described below.

[0219] AAV production in the HEK293 transient transfection system was assessed by transfecting HEK293 cells with three plasmid DNAs including pAdhelper (including E2A, E4, L4 and VA RNAs), pAAV-Rep / Cap / Transgene, and pEF1a-L4 constructs or pUC57 stuffer control. The plasmids were mixed at a 1 :1 :1 ratio and transfected as described above. Transfected cultures were collected at 3 days post-transfection for analysis of rAAV replication and production as described below.

[0220] Impact of supplementing 22K, 33K into pAdhelper on AAV production was also assessed under the HEK293 transient transfection platform condition. EF1a promoter driven L4-22K or L4-33K native sequence or codon-optimized sequence were cloned into pAdhelper plasmids resulting in pAdH-22K-WT, pAdH-33K-WT, pAdH-22K-CO, pAdH-33K-CO. Supplementing 22K and 33K as a separate plasmid were included as a positive control. pUC57 was used as DNA vehicle control for equal total DNA mass. TPP and pAdhelper ratio of 1 :1 and 1 :3 was used for TPP-1 ; 1 :1 only was used for TPP-2. AAV productivity was analyzed using 72 hours post transfection harvest via real-time qPCR, as described below.

[0221] AAV replication and production were analyzed as previously described (Martin, et al., 2013). Briefly, whole production cultures were collected and lysed with TWEEN®-20 (0.5%) followed by treatment with Benzonase (EMD, McCook, IL, cat# 1.01697.0001 , 10U / ml) for AAV production titer analysis. Benzonase treated cell lysates were further digested with DNase (Promega, Madison, Wl, cat# M610A; 60 ll / rnl final concentration) for 10 min at 37°C. The DNase reaction was terminated via addition of Proteinase K (Invitrogen, Waltham, MA, cat# 100005393; 0.5 mg / mL final concentration) and incubation at 65°C for 10 min followed by heat inactivation at 95°C for 20 min. Heat-inactivated post Proteinase K samples were then diluted and analyzed with QuantStudio 7flex using a BGH polyA primer / probe set listed in Table 1 following standard qPCR procedures. AAV genome copy numbers were calculated via comparison to a standard curve generated with a plasmid (pAF196) that contained all PCR target sequences (e.g., rep, cap, BGH polyA, E2A). For analysis of total DNA copies, the same procedure was followed without Benzonase or DNase treatment.

[0222] Analysis of adenovirus production and replication

[0223] Adenovirus production was quantified as DNase-resistant genomes per mL, employing a methodology akin to the AAV titer analysis described above, but utilizing the E2A primer / probe set from Table 1 in accordance with standard qPCR protocols using QuantStudio7flex. For assessing adenoviral replication, total genome copies per mL were measured using the same protocol, but with the exclusion of Benzonase and DNase treatment.

[0224] Rep / cap gene amplification analysis

[0225] Rep / cap genes stably integrated in producer cell lines undergo amplification after wtAd5 infection. The amplified copies exist as extrachromosomal DNA (ecDNA) (Tessier, et al. 2001 ; Liu, etal. 2000). To assess rep / cap amplification, ecDNA was extracted from infected producer cell pellets using Purelink Hipure DNA extraction mini kit (Life technology, Waltham, MA, cat# K210003). To quantify rep / cap amplification, duplex dPCR using rep / cap specific primer / probes (Table 1) were performed using ecDNA as the template. The dPCR was carried out with QIAcuity Probe PCR Kit (Qiagen, Germantown, MD, cat# 250102) according to the manufacturer’s protocol on a QIAcuity 8. rep / cap gene copies were normalized to the cell number for comparison as described previously (Liu, et al., 2000).

[0226] AA V transcription analysis via Northern blot

[0227] Total RNA was isolated from the wtAd5-infected producer cell line using RNeasy plus kit (Qiagen, Germantown, MD, cat# 74134) according to manufacturer’s instructions and used for further analysis. 1 to 2 mg total RNA from each sample was run and transferred to a Nylon membrane (Roche, Basel, Switzerland, cat# 11209299001) using the NorthernMax-Gly kit (Invitrogen, Waltham, MA, cat# AM1946) according to the manufacturer’s protocol. A digoxigenin (DIG)-labeled probe targeted the cap sequence was prepared using the PCR DIG probe Synthesis kit (Roche, Basel, Switzerland, cat# 11636090910) according to the manufacturer’s instructions. The forward primer sequence was 5’- TGCAGGCGGGTGACAAT-3’ (SEQ ID NO: 43) and the reverse primer sequence was 5’- GGTTGTCGTTGCTGGCCCC-3’ (SEQ ID NO: 44). The blot was UV-crosslinked and prehybridized in EasyHyb solution (Roche, Basel, Switzerland, cat# 11603558001) for 30 min before hybridization with denatured probe at 55°C overnight. The blot was then washed and developed using the DIG Wash and Block buffer set (Roche, Basel, Switzerland, cat# 11585762001) and DIG luminescent Detection kit (Roche, Basel, Switzerland, cat# 11363514910), according to the manufacturer’s protocol. Blots were visualized and imaged with the BioRad Chemidoc MP imaging system. The blot image was further quantified using BioRad Image Lab Software v6.1.0.

[0228] Transcription and A A V splicing analysis via RT-dPCR

[0229] For quantification of rep / cap transcripts, total RNA isolated from infected producer cells or transfected HEK293 cells was reverse transcribed to cDNA using SuperScript™ IV VILO Master Mix with ezDNase™ Enzyme (Invitrogen, Waltham, MA, cat# 11766050) followed by quantification via dPCR using QIAcuity Probe PCR Kit (Qiagen, Germantown, MD, cat# 250102) on a QIAcutiy 8. Serial dilutions of cDNA reaction were used as the template for dPCR using primers / probes targeting total rep, rep / cap transcripts or spliced rep / captranscripts (FIG. 20, right and Table 1). GAPDH was used as the internal loading control. Relative rep or rep+cap total mRNA was calculated from normalized dPCR values compared against the wtAd5 infection condition at MOI of 5, non-targeting DsiRNA negative control or pUC57 control. AAV rep / cap splicing efficiency was assessed by dividing dPCR values obtained using primers / probe (splicing set) specifically targeting spliced transcripts by values using primers / probe targeting total rep / cap transcripts (rep / cap set). Adenoviral mRNA was analyzed with QIAcuity EG PCR kit (Qiagen, Germantown, MD, cat# 250112) with targetspecific primers (Table 1) and then normalized to GAPDH as the internal control.

[0230] Western blot analysisCell pellets were lysed in RIPA lysis and extraction buffer (Life technology, Waltham, MA, cat# 89900) supplemented with Benzonase (EMD, McCook, IL, cat# 1.01697.0001 , 10U / mL), MgCI2 (2mM) and Halt protease inhibitor cocktail (Thermo, Waltham MA, cat# 78425). Total cell lysates were subjected to electrophoresis in a 4-12% NuPAGE Bis-Tris gel in NuPAGE MOPS running buffer (Invitrogen, Waltham, MA, cat# NP0001) and then transferred to a nitrocellulose membrane (Invitrogen, Waltham, MA, cat# IB23001)) using the iBlot2 dry blotting system. The primary antibodies used were anti-AAV2 Rep (Fitzgerald, Gardner, MA, cat# 10RA140a, 1 :200), anti-AAV2 Cap (American Research Products, Atlantis, FL, clone B1 , cat# 690058, 1 :1000), anti-p-tubulin Dylight 680 (Invitrogen, Waltham, MA, cat# NA516308D680, 1 :2000), anti-Ad DBP (CUSABIO, Houston, TX, cat# CSB- PA365892ZA01 HIL, 1 :2000), Anti-Ad E1A (Santa Cruz, Dallas, TX, cat# sc-25, 1 :2000), and anti-Ad5 capsid (Abeam, Waltham, MA, cat# AB6982, 1 :10000). Goat-anti-mouse H+L HRP (Bio-Rad, Hercules, CA, cat# 1706516, 1 :3000) secondary antibody was used for anti-Rep and anti-Cap; Goat anti-Rabbit IgG (H+L) Secondary antibody, HRP (Invitrogen, Waltham, MA, cat# 32460, 1 :2000) was used for anti-Ad DBP, anti-Ad E1A; Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, DyLight 800 (Invitrogen, Waltham, MA, cat# SA510036) was used for anti-Ad5 capsid. The ECL (Bio-Rad, Hercules, CA, cat# 1705060) was used for detection and blot was imaged with BioRad ChemiDoc MP imaging system. The blot image was further quantified using BioRad Image Lab Software v6.1.0.Results

[0231] I. Suppression of Adenovirus replication with the adenovirus variant Ad5ts149 reduces AAV vector production.

[0232] The AAV producer cell line platform requires infection with wtAd5 to induce rAAV production, a process in which the adenoviral helper is co-replicated and produces viral progeny in conjunction with the AAV vector. In this example, the effect on production of using Ad5ts149, a temperature-sensitive mutant, to provide helper function in place of wtAd5 was evaluated. PCL cells were infected with wtAd5 or Ad5ts149 at either the semi-permissive(37°C) or restrictive (39°C) temperature. Production of rAAV and adenovirus was quantified as Nuclease-resistant vector genome copies per mL using culture samples treated with nuclease as the template for qPCR as described in Material and Methods. Replication of rAAV and adenovirus were measured as total viral genome copies per mL in a similar manner except nuclease treatment was omitted in the sample processing procedure. Results from different conditions were normalized to that of wtAd5 infection with MOI of 5 at 37°C. As expected, at the semi-permissive temperature, replication and production of Ad5ts149 was reduced by 70- 80% relative to wtAd5 while at the restrictive temperature, Ad5ts149 replication and production was two- to three-logs lower than at 37°C (FIGs. 7A-B and Table 2). Increasing the MOI at 39°C resulted in an apparent dose-dependent increase in both replication and production of Ad5ts149. In contrast, with wtAd5 infection only a slight decrease in replication and production was observed at 39°C relative to 37°C.Table 2: Mean (SD) of the relative ratio of AAV and Ad production and replication from wtAd5 and Ad5ts149 comparison study in Figure 7.Shown are the averages (standard deviation) from three or four biological replicates. The relative ratios of AAV and Ad production and replication were calculated by normalizing to the values of the condition of wtAd5, MOI=5 at 37°C from each independent run of the three replicates.

[0233] Ad5ts149 supports rAAV production at a level comparable to wtAd5 at 37°C, despite the four to five- fold reduction in adenoviral production and replication at the semi-permissive temperature (FIGs. 7C-D, and Table 2). However, at the restrictive temperature of 39°C, production of rAAV with Ad5ts149 was significantly reduced. At MOI=4, rAAV titer was 70-fold lower than that of wtAd5 infection at 37°C (FIG. 7C). As was observed with the Ad5ts149 helper virus, at 39°C rAAV exhibits an apparent dose-dependent increase in production with increasing MOI of Ad5ts149. In contrast, only a four-fold reduction in replication of the AAVvector genome was observed at the lowest MOI of Ad5ts149 at 39°C (FIG. 7D). Overall, these results suggest that rAAV vector production from producer cells is dependent upon efficient replication and production of the helper virus. Notably, however, when replication of the helper virus was limited {e.g., Ad5ts149, MOI=4 at 39°C) rAAV genome replication was largely maintained. Therefore, other features of vector production, such as AAV capsid expression and / or genome packaging, likely explain the drop in rAAV production under these conditions.

[0234] II. Suppression of Adenovirus replication and production altered AAV rep / cap transcription, splicing and protein expression.

[0235] To address why rAAV production was reduced with Ad5ts149 infection at the restrictive temperature, AAV Rep and Cap protein expression were evaluated. While Cap protein expression in wtAd5 infected cells was similar at 37°C and 39°C, Ad5ts149 infection at 39°C resulted in significantly lower expression of Cap protein (FIGs. 8A-B) . Similarly, while Rep expression remained similar overall for the wtAd5 infection at the two temperatures, a different pattern of Rep expression emerged following infection with Ad5ts149 (FIG. 8C-G). At 37°C, an increase in expression of both Rep78 and Rep68 compared to wtAd5 was observed, with the increase in Rep68 being particularly notable. When Rep protein expression in Ad5ts149-infected cells was compared at the two temperatures, a clear reduction in expression of the Rep68 and Rep40 proteins (which are derived from spliced transcripts) at 39°C in addition to a slight increase in the expression of the Rep78 and Rep52 proteins (which are derived from unspliced transcripts) were observed. Increasing the MOI of Ad5ts149 at 39°C enhanced Cap, Rep68, and Rep40 proteins expression in a dose-dependent manner. Taken together, these results provide evidence for a correlation between expression of the AAV Rep68 / 40 and Cap proteins and AAV vector production in the context of an Ad5ts149 infection at the restrictive temperature. The data suggest a dependence of Rep68 / 40 and Cap protein expression on adenoviral factors that are lacking in the absence of viral replication and production, presumably the adenoviral late gene products.

[0236] Amplification of the AAV rep and cap genes is critical for efficient AAV production from stable producer cell lines (Chadeuf, et al., 2000; Tessier, et al., 2001 ; Nony, et al., 2001 ; Gao, et al., 2002). In this example, rep gene amplification by dPCR was assessed (FIG. 8H) and the results show that rep gene copy numbers were largely comparable for Ad5ts149 and wtAd5 at both temperatures (Table 3). Similar results were obtained for the cap gene (Table 3). However, with wtAd5 infection at the MOI of 100, rep / cap copy numbers seemed to be reduced compared to infections at lower MOIs. This may be a result of competition from adenoviral replication at the high MOI, but the specific mechanism requires further investigation. These results imply that the reduction in rAAV vector production from Ad5ts149 at the restrictive temperature is not linked to a decrease in rep / cap amplification. Next, rep / capgene expression at the transcriptional level was assessed by Northern blot (FIG. 81). The pattern of AAV transcripts was uniform at 37°C and 39°C for wtAd5. Ad5ts149 infection at 37°C showed a similar pattern, but at the restrictive temperature of 39°C the overall rep / cap transcription level and the relative abundance of the six AAV transcripts were altered. The Northern blot revealed an increase in the level of the unspliced p5 (4.2 kb) and p19 (3.6 kb) rep gene transcripts as well as the 2.6 kb unspliced p40 transcript. This increase was accompanied by a reduction in the corresponding spliced transcripts. This altered pattern of AAV transcript abundance is consistent with the reduction of expression of the AAV Rep68, Rep40, and Cap proteins which are translated from spliced transcripts (FIG. 8A and 8C).Table 3: Raw data of rep / cap amplification evaluated from three biological replicates used in FIG. 8B.

[0237] To confirm the Northern blot results, AAV transcripts were quantified by RT-dPCR. The level of rep mRNAs trended higher for Ad5ts149 at 37°C compared to wtAd5 (FIG. 8I-J). This result is consistent with the results of the Northern blot and with the observed increase in Rep (especially Rep68) protein level at 37°C (FIGs. 8C and 8H). At 39°C the mRNA levels of rep and total rep / cap in Ad5ts149-infected cells exhibited a dose-dependent pattern: at an MOI of 100, the levels were similar to those at 37°C, but at an MOI of 4, the abundance was reduced five to ten-fold (FIG. 8J-K). This RT-dPCR result is also consistent with the pattern observed on the Northern blot (FIG. 8I). To assess the efficiency of splicing of AAV rep and cap transcripts, RT-dPCR was performed using specific primers and probes capable of differentiating the spliced and unspliced transcripts (FIG. 2C, right). For wtAd5, approximately 75% of the total replcap transcripts were spliced, regardless of the infection temperature (FIG. 8L). Ad5ts149 infection at 37°C produced similar levels of splicing. However, at 39°C a two to three-fold reduction in splicing with Ad5ts149 infection at MOIs of 4 and 15, respectively, was observed. Consistent with Rep and Cap protein expression and AAV vector production at 39°C, AAV splicing showed a trend toward a dose-dependent increase with MOI, suggesting that a common limiting factor associated with Ad5ts149 infection at the restrictive temperature mightexplain the altered AAV transcript splicing pattern and subsequent deficit in rAAV production with this helper virus.

[0238] III. Adenovirus late gene expression is limited with Ad5ts149 infection at the restrictive temperature.

[0239] To identify the viral factors that might be involved in the splicing of AAV transcripts, the expression of adenoviral helper genes was analyzed by RT-dPCR. The levels of mRNAs derived from the adenoviral early genes E1A and E2A with Ad5ts149 infection were comparable to or higher than wtAd5 at both 37°C and 39°C (FIGs. 9A-B; and results from analysis of additional early genes are shown in FIG. 13A-E). Analysis of E1A and E2A protein expression via Western blot (FIG. 9C) was consistent with the RT-dPCR results, showing equivalent (E2A) or higher (E1A) protein expression from Ad5ts149 at 39°C compared to wtAd5. In contrast, but as expected, hexon mRNA was significantly reduced with Ad5ts149 infection at the restrictive temperature (FIG. 9D). Using an anti-Ad5 capsid antibody, this study also evaluated the expression of late structural proteins via Western blot (FIG. 9E). A significant reduction in expression of the late gene products was observed with Ad5ts149 infection at 39°C compared to 37°C or to wtAd5 infection at either temperature; however, there was a dose-dependent increase in these products with Ad5ts149 infection at 39°C. In summary, expression of late gene products is compromised with Ad5ts149 infection at the restrictive temperature, while early gene products are unaffected, suggesting that AAV splicing is likely regulated by adenoviral late, and not early gene products.

[0240] The adenovirus major late transcription unit encodes multiple late proteins from five regions, L1 , L2, L3, L4 and L5 (FIG. 9F). Since the L4-22K / 33K gene products have been reported to regulate splicing of adenoviral transcripts, their role in regulating AAV splicing was examined (Morris, et al., 2009; Tdrmanen, et al., 2006; Guimet, et al., 2013; Biasiotto, et al., 2015). Using RT-dPCR, L4-22K / 33K gene expression following infection with wtAd5 or Ad5ts149 was compared (FIG. 9G-I). When compared to infection at 37°C, infection of Ad5ts149 at 39°C resulted in a significant reduction in expression of the L4 genes encoding the 100K, 22K, and 33K proteins. Expression showed a dose-dependent increase with increasing MOI. At 39°C with an MOI of 4, abundance of L4 mRNAs was reduced 10 to 100- fold relative to 37°C, whereas at an MOI of 100, levels were comparable at the two temperatures.

[0241] IV. Adenovirus L4 genes regulate rep / cap splicing and expression.

[0242] To assess whether reduced L4 gene expression might explain the deficit in AAV splicing observed with Ad5ts149 infection at 39°C, dicer-substrate siRNAs (DsiRNA) was usedto knock down L4 gene expression (see FIG. 4A and 9F; as described by Su et al. (2023)) in a PCL infected with wtAd5. Greater than 70% knockdown of L4-100K / 22K / 33K gene (also referred to as “L4-100 / 22 / 33K”) expression was achieved using the 100K-specific DsiRNAs while 22K / 33K and L4 A-targeted DsiRNAs achieved over 90% knockdown (FIG. 10A). This level of knockdown efficiency likely reflects the fact that the 100K A / B DsiRNAs are specific for the 100K gene, while the 22K / 33K and L4-A DsiRNAs target all three L4 genes (FIG. 14A- B). Knockdown of L4 gene expression reduced AAV production significantly (FIG. 10B). The L4-22K / 33K DsiRNA had the greatest impact, reducing AAV production by 50 to 100-fold compared to the DsiRNA negative control. Knockdown of these genes also reduced rep DNA copies by 20-fold (FIG. 10C, and Table 4) while treatment with the L4-100 K- targeted DsiRNAs resulted in a 10-fold reduction. In addition, AAV rep / cap mRNAs (FIG. 10D) were reduced by approximately 50-fold with the L4-22K / 33K and L4 A DsiRNAs and by approximately 3-fold with L4-100K knockdown. The RT-dPCR results were consistent with the pattern observed in the Northern analysis (FIG. 10E). Densitometric quantitation of the cap transcripts revealed that the ratio of the 2.3kb spliced to the 2.6kb unspliced transcript decreased from approximately seven in the negative control to two for all L4 DsiRNAs (FIG. 10F). Furthermore, RT-dPCR confirmed a reduction in AAV transcript splicing from 75% to approximately 50% by all L4 DsiRNAs (FIG. 10G), supporting the hypothesis that L4 gene products play a role in AAV splicing. These results are supported by the western analysis, which showed that the L4- 22K / 33K and L4 A DsiRNAs reduced levels of both Rep and Cap proteins (FIG. 10H). Overall, rep / cap gene amplification, transcription, and protein expression consistently reflected the level of AAV production observed upon knockdown of each of the L4 genes. Taken together, these results suggest that the adenovirus L4 gene products play an essential role in AAV gene expression and virus production, as they regulate not only rep and cap gene amplification as previously reported (Su, et al., 2023), but also the synthesis and splicing of AAV transcripts.

[0243] Table 4: Raw data table for rep / cap amplification evaluation from three biological replicates used in Figure 10C.

[0244] V: Supplementation of the L4-33K gene improves AAV splicing and vector production.

[0245] T o further establish the role of the adenoviral L4 genes in the regulation of AAV gene expression, the L4 genes were supplemented in the context of an Ad5ts149 infection at 39°. Without being bound by theory, enhancing L4-22K / 33K gene expression was hypothesized to result in improved rep / cap splicing and gene expression. Plasmid constructs expressing the individual L4 genes, 100K, 22K, 33K or pVIII from the EF1a promoter were transfected into producer cells. The next day, the cells were infected with Ad5ts149 at 39°C. AAV gene expression, replcap transcript splicing, and vector production were subsequently analyzed. Supplementation of either the L4-22K or L4-33K gene but not the 100K or pVIII genes enhanced AAV production by two-fold (FIG. 11 A). To validate this hypothesis, the levels of rep / cap mRNA were quantified by RT-dPCR. Supplementation of L4-22K was associated with an increase in AAV rep / cap mRNA relative to the control (FIG. 11B). However, only supplementation of the L4-33K gene increased splicing efficiency relative to the negative control (from -26% to -38%; FIG. 11C). Consistent with this result, the abundance of AAV Cap and the Rep68 / Rep40 proteins also showed an increase with supplementation of L4-33K (FIG. 11D). As expected, rep / cap gene amplification was not affected by supplementation of any of the L4 genes (FIG. 15 and Table 5). Supplementation of 100K or pVIII via transfection of the EF1a-100K or pVIII construct did not impact either splicing or mRNA levels, although DsiRNA-mediated knockdown of the 100K gene resulted in a reduction in the level of AAV splicing in the context of a wtAd5 infection (FIG. 10F and G).

[0246] Taken together, these results indicate that Adenovirus L4 gene products regulate AAV Rep / Cap expression and production at multiple levels. Adenovirus L4-33K regulates splicing of AAV transcripts whereas L4-22K seems to regulate transcript abundance. L4-22K is a transcriptional regulator essential for the transition from early to late adenoviral gene expression (Morris, et al., 2009; Lan, et al., 2017; Wu, et al, 2012). Therefore, the L4-22K gene product may directly or indirectly regulate transcription of AAV genes. The observed reduction in AAV splicing from 100K knockdown could result from an indirect effect on expression of the L4-22K and L4-33K proteins since 100K is required for efficient adenoviral late mRNA translation and deletion of 100K significantly reduced expression of some late genes (Hayes, et al., 1990; Hodges, et al., 2001). Full restoration of AAV productivity from Ad5ts149 infection at 39°C may require near wild-type levels of L4-33K as well as the other L4 genes.Table 5: Raw data table for rep / cap amplification evaluation from three biological replicates used in Figure 15.

[0247] VI. L4-33K regulates AAV rep / cap splicing and impacts AAV vector production in HEK293 cells.

[0248] Given the role of the L4-22K / 33K gene products in AAV gene expression and vector production in the context of producer cells, this study evaluated supplementation of the L4 genes in the HEK293 transient transfection platform. Supplementation of L4-33K, in addition to the intrinsic L4 gene expression from the pAdhelper plasmid, led to a productivity increase for multiple AAV vectors expressing different transgenes and capsid types (FIG. 12A). Analysis of AAV CapdGene4 transcript splicing revealed that, similar to the effect in the PCL, supplementation of the L4-33K gene resulted in an increase in splicing efficiency from -50% to -75% (FIG. 12B). These results suggest that supplementation of L4-33K might be a broadly applicable strategy to improve vector production in the HEK293-based transfection system. As shown in FIGs. 12A-B, supplementing L4-33K as a separate plasmid consistently supported 2- to 3-fold higher AAV production. Supplementing L4-33K via cloning into pAdhelper (pAdhelper-33K) however also resulted in about 2-fold AAV productivity increase at Triple-play plasmid (TPP) to pAdhelper ratio of 1 :1 for both TPP-1 and TTP-2, but less impact is observed at a ratio of 1 :3. (FIG. 12C).

[0249] Discussion

[0250] Generation of recombinant AAV in an AAV producer cell involves a complex interaction between the helper adenovirus and the viral vector. For example, the five adenoviral helper factors, E1A, E1 B, E2A, E4orf6 and VA RNAs which are necessary to support AAV gene expression and viral genome replication are also required to support these processes for wtAd5, leading to competition between the helper and the AAV. In addition, the wtAd5 helper and AAV also vie for availability of cellular resources, e.g., the nucleotides, amino acids and energy required for virus production. To mitigate competition and promote its own proliferation, AAV has evolved strategies, such as Rep-mediated repression of adenoviral gene expression, which interfere with replication of the helper virus (Timpe, et al., 2006). This repression is accomplished directly via binding of Rep to viral promoters, or indirectly via inhibition of the cAMP-dependent protein kinase A (PKA) pathway (Casper, et al., 2005; Needham, et al., 2006; Pasquale, et al., 2003).

[0251] In this study, Ad5ts149, an adenovirus mutant with diminished replication due to a temperature-sensitive defect in the DNA polymerase gene, was evaluated as a helper virus inthe context of a model AAV producer cell line. The objective was to reduce replication and production of adenovirus in the producer platform, and to understand more fully the interplay between the helper adenovirus and AAV during vector production. Utilizing this model producer cell line as an example, the results above showed that at the semi-permissive temperature of 37°C, Ad5ts149 titer was reduced by about 70% to 80% relative to wtAd5. However, at this temperature, Ad5ts149 infection supported AAV production at a level comparable to wtAd5 (FIG. 7). This finding is consistent with the results of Farson et al., (2004) who observed a half to a log reduction in Ad5ts149 titer at 37°C following infection of an A549- based AAV producer cell line. At this temperature, production of AAV with Ad5ts149 was similar to that observed with Add / 309, a replication-competent E3-deleted virus.

[0252] At the restrictive temperature of 39°C, where Ad5ts149 replication was reduced by two to three logs, AAV vector production was significantly impaired. Further investigation revealed that under these conditions, AAV Cap protein expression was significantly reduced. This was accompanied by a selective decrease in expression of the Rep68 and Rep40 proteins and consequently, an alteration of the pattern of Rep protein expression (FIG. 8A and B. Notably, an increase of the MOI from 4 to 100 increased replication of Ad5ts149 at 39°C and coincided with a dose-dependent increase in the expression of the Rep68 / 40 and Cap proteins and AAV productivity (FIG. 8J-L). Ad5ts149 infection at the low MOI reduced AAV vector genome replication only slightly (~two-fold, FIG. 7D). The maintenance of AAV vector genome replication at the restrictive temperature is likely explained by expression of Rep78, which is equal to or greater than expression at 37°C (FIG. 8C and D). Continued expression of Rep78 also likely explains the maintenance of rep / cap amplification with Ad5ts149 infection at 39°C. Like Rep78, expression of E1A was also enhanced at 39°C in Ad5ts149-infected cells (FIG. 9C). Since E1A activates the AAV p5 promoter, this enhanced expression may explain the high level of Rep78 protein expression that is observed at the restrictive temperature (Chang, et al., 1989).

[0253] Splicing of AAV transcripts is required for expression of Rep68 and Rep40 and for expression of the capsid proteins VP1 , VP2 and VP3. Use of alternate splice acceptor sites located at nucleotides 2201 (minor splice acceptor) and 2228 (major splice acceptor) in the AAV2 genome helps to establish the correct stoichiometry of the three capsid proteins (Laughlin, et al., 1982; Qiu, et al., 2008; Trempe, et al., 1988; Becerra, et al., 1988). Ad5ts149 infection at 39°C causes a reduction in Rep68, Rep40, and all three capsid proteins (FIG. 8A to G) This pattern correlates with a potential splicing defect at this temperature, which was confirmed by Northern analysis and RT-dPCR (FIG. 8I and L). Consistent with previous reports (Mouw and Pintel, 2000), at 37°C, spliced transcripts (2.3kb) made up the majority of the p40 transcripts from either wtAd5 or Adt5s149 infection; however, spliced p5 and p19 transcripts were also present at a higher level than unspliced transcript (Mouw and Pintel,2000). The AAV transcript patterns are similar for wtAd5 at 37°C and 39°C and Ad5ts149 at 37°C, but Ad5ts149 infection at 39°C leads to a general shift towards more unspliced transcripts. Quantitation via RT-dPCR was consistent with Northern blot analysis: -75% of transcripts were spliced with wtAd5 infection at 37°C but a relative reduction in splicing from 75% with wtAd5 to 20-40% was observed with Ad5ts149 at 39°C (FIG. 8K). The percentage of spliced transcripts for wtAd5 (-75%) is consistent with Mouw and Pintel (2000) but higher than that reported by Stutika et al. (2016). Different analytical methods and splicing variant coverage likely contribute to this difference. Overall, these results suggest that in the absence of efficient replication of the helper viral genome, one or more adenoviral factor(s) is likely limiting, resulting in the observed AAV splicing defect in the producer cell line platform. Adenovirus infection is known to stimulate splicing of AAV transcripts (Mouw and Pintel, 2000), however the adenoviral gene product(s) involved in this process have yet to be identified.

[0254] The dramatic effect on Rep and Cap protein expression and splicing of AAV transcripts resulting from defective replication of the Ad5ts149 helper prompted us to hypothesize that an adenoviral late gene product(s) was required for replication and packaging of the rAAV. As expected, adenoviral late gene expression was reduced at 39°C (FIG. 9D-I) and abundance of the adenoviral structural proteins and transcripts derived from the L4 region correlated with the level of replication of Ad5ts149. DsiRNA-mediated knockdown of the adenoviral late genes, L4-22K and L4-33K confirmed that products of both genes are required for vector production in the PCL.

[0255] Although it was previously thought that the minimal set of adenoviral helper functions were largely early genes, novel roles for the L4-22K and L4-33K gene products in AAV vector production have recently been described (Adsero, et al., 2023). L4 genes are expressed early in the late phase from the L4 promoter (L4P), but due to negative feedback regulation of L4P by L4-22K and L4-33K, transcription during the late phase ultimately comes under the control of the stronger major late promoter (MLP) (Wright, etal., 2015). L4-22K and L4-33K are related proteins that share an N-terminal sequence; however, because the L4-33K gene product is translated from a spliced transcript, the proteins differ in their carboxyl-terminal domains (Biasiotto, et al., 2015). The L4-22K and L4-33K gene products are multifunctional and have been shown to play a role in adenoviral genome packaging as well as in regulation of the transition from early to late gene expression (Morris, et al., 2009; Wu, et al., 2012; Backstrdm, et al., 2010; Wu, et al., 2013). L4-33K acts as a virally encoded splicing factor to regulate alternative splicing of the adenoviral major late transcripts (Tdrmanen, et al., 2006; Wu, et al., 2013; Farley, et al., 2004). In this role, L4-33K specifically promotes the use of weak 3’ acceptors that are believed to bind the splicing factor LI2AF inefficiently due to the absence of long pyrimidine tracts characteristic of consensus acceptor sites (Muhlemann, et al., 1995). Notably, the sequences of both the minor and major 3’ splice acceptor sites of AAV lackextended pyrimidine tracts, and therefore may represent potential targets of splicing activation by L4-33K. Interestingly, Mouw and Pintel (2000) reported a shift in the abundance of spliced AAV transcripts in HEK293 cells coinfected with AAV and adenovirus when the cells were preinfected for 12 hours with adenovirus. This observation is consistent with the involvement of a late gene product in AAV splicing. As shown here, supplementation of L4-33K in the context of an Ad5ts149 infection at 39°C was able to increase Cap protein expression and AAV production while splicing of rep / cap transcripts was enhanced from 26 to 38 percent (FIG. 11 A, C, and D). This relatively small increase in splicing may be attributed to a relatively low level of expression of L4-33K following transient transfection of the PCLwith the EF1a-33K plasmid. RT-dPCR analysis showed that expression from the supplemented L4 gene was more than one log lower than that observed following infection with wtAd5 (FIGs. 17A-D). This low level of expression of L4-33K might also explain why the rAAV productivity was increased only twofold with supplementation and was not fully rescued. Taken together, these results demonstrate a novel role for L4-33K in AAV vector production.

[0256] Interestingly, the L4-22K gene product did not seem to have an impact on splicing of AAV transcripts, but supplementation of L4-22K in cells infected with Ad5ts149 at 39°C increased AAV production along with rep / cap gene transcription (FIG. 11A and B). This raises the possibility that L4-22K may regulate the level of rep / cap transcripts either by altering promoter activity or via an effect on mRNA stability. During the adenoviral life cycle, L4-22K has been proposed to both activate transcription of the MLP (Backstrbm et al., 2010) and facilitate post-transcriptional processing of viral mRNAs (Morris, et al., 2009). L4-22K has also been reported to suppress early gene expression during the late phase of infection; L4-22K mutant viruses show increased E1A expression (Wu, et al., 2012). Therefore, reduced expression of the L4-22K gene product may at least partially explain the increase in E1A protein observed with Ad5ts149 infection at 39°C (FIG. 9C).

[0257] Involvement of L4-22K and L4-33K in AAV production was recently demonstrated by using a novel helper virus (TESSA-E1) that is replication-competent but contains a selfrepressing MLP (Su, et al., 2023). Repression of the MLP interferes with late gene expression and consequently with adenoviral packaging. However, when TESSA-E1 was used as a helper in the HeLaRC32 AAV packaging cell line, amplification of the integrated AAV rep / cap genes was reduced. In the study performed by Su et al. (2023) knockdown and trans supplementation experiments confirmed that the L4-22K and L4-33K genes play a role in rep / cap gene amplification. The authors hypothesized that reduced amplification was due to sequestration of the L4-22K / 33K gene products bound to replicating TESSA-E1 genomes and subsequently demonstrated that the amount of L4-22K / 33K produced without MLP activation was sufficient to support rep / cap amplification when adenoviral replication was inhibited. In the current study, expression of the L4 100K / 22K / 33K genes was reduced in cells infectedwith Ad5ts149 at the restrictive temperature (FIG. 9G-I), but amplification of the rep gene was unaffected (FIG. 8H, Table 3). Since replication of Ad5ts149 is reduced at the restrictive temperature, this observation is consistent with the hypothesis that replicating adenoviral genomes sequester the low levels of L4-22K / 33K expressed from the L4P promoter (Id). A separate study reported that a 100K-deleted adenovirus was unable to support rep / cap amplification in a stable AAV2 producer cell line, whereas amplification was unaffected when a replication-defective DpTP mutant was used as a helper (Kruger-Haag, et al., 2019). In agreement with this published study, knockdown of L4-100K in the context of a wtAd5 infection resulted in a 10-fold decrease in rep DNA copies, thereby reducing Rep and Cap protein expression. In addition, a 20-fold reduction in rep DNA copies was observed with knockdown of L4-22K / 33K (FIG. 10C, left). These results confirm previous reports and demonstrate that the L4 gene products play a role in amplification of rep / cap gene sequences in AAV producer cell lines.

[0258] In the HEK293 transient transfection platform, Adsero et al. (2024) showed that the L4-22K gene product was essential for rAAV production, while co-expression of L4-33K contributed to a boost in productivity (Adsero, et al., 2023). In those studies, deletions were introduced in the adenovirus helper plasmid encoding the E2A, E4 and VA RNAs. Deletions encompassing the E2A promoter which overlaps the L4 gene region resulted in reduced AAV vector titer. Rescue of L4-22K or L4-33K expression either via insertion into the non-productive adenovirus helper plasmid or in trans via transfection of a separate plasmid recovered productivity. In contrast, in the studies described here, a plasmid expressing either the L4-22K, L4-33K or additional L4 genes was co-transfected into HEK293 cells in conjunction with an intact and functional adenovirus helper plasmid. Increased expression of L4-33K but not L4- 22K or other L4 genes via this supplementation strategy resulted in a two to three-fold increase in AAV productivity for multiple AAV vectors (FIG. 12A). RT-dPCR analysis confirmed that in this platform as well, L4-33K appears to enhance splicing of AAV transcripts (FIG. 12B). Therefore, the L4-33K gene product is an important factor contributing to AAV production across different vector production platforms. A reduction of AAV splicing and production resulting from 100K supplementation in this experiment differs from the observation in the PCL platform, indicating potentially unique mechanisms of regulation of AAV transcription splicing and production by the L4 genes in the transient transfection platform. Further investigation would be required to delineate the specific differences.

[0259] Overall, these results advance the understanding of adenoviral helper function in the AAV production process, highlighting L4-22K and L4-33K as adenoviral helper genes for robust vector production in the AAV producer cell system, and revealing a novel role for L4- 33K in the splicing of AAV transcripts.References (Example 7)1. Au, H.K.E., Isalan, M., and Mielcarek, M. (2022). Gene Therapy Advances: A MetaAnalysis of AAV Usage in Clinical Settings. Front. Med. 8, 809118.2. Zhao, Z., Anselmo, A.C., and Mitragotri, S. (2022). Viral vector - based gene therapies in the clinic. Bioeng. Transl. Med. 7, e10258.3. Xiao, X., Li, J., and Samulski, R.J. (1998). Production of High-Titer Recombinant Adeno- Associated Virus Vectors in the Absence of Helper Adenovirus. J. Virol. 72, 2224-2232.4. Grimm, D., Kern, A., Rittner, K., and Kleinschmidt, J. A. (1998). Novel Tools for Production and Purification of Recombinant Adenoassociated Virus Vectors. Hum. Gene Ther. 9, 2745-2760.5. 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Novel cis -Acting Replication Element in the Adeno-Associated Virus Type 2 Genome Is Involved in Amplification of Integrated rep-cap Sequences. J. Virol. 75, 9991-9994.Gao, G., Lu, F., Sanmiguel, J.C., Tran, P.T., Abbas, Z., Lynd, K.S., Marsh, J., Spinner, N.B., and Wilson, J.M. (2002). Rep / Cap Gene Amplification and High-Yield Production of AAV in an A549 Cell Line Expressing Rep / Cap. Mol. Ther. 5, 644-649. Hayes, B.W., Telling, G.C., Myat, M.M., Williams, J.F., and Flint, S.J. (1990). The adenovirus L4 100-kilodalton protein is necessary for efficient translation of viral late mRNA species. J. Virol. 64, 2732-2742. Hodges, B.L., Evans, H.K., Everett, R.S., Ding, E.Y., Serra, D., and Amalfitano, A. (2001). Adenovirus Vectors with the 100K Gene Deleted and Their Potential for Multiple Gene Therapy Applications. J. Virol. 75, 5913-5920. Timpe, J.M., Verrill, K.C., and Trempe, J.P. (2006). 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[0260] Those skilled in the art will recognize, or will be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments, described herein. The scope of the present disclosure is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

[0261] In the claims articles such as “a,” “an,” and “the” can mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptionsthat include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0262] Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and / or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

[0263] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the methods, compositions, and systems described herein is / are referred to as comprising particular elements, features, etc., certain embodiments or aspects of the methods, compositions, and systems consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments, described herein. The scope of the present disclosure is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

[0264] By “comprising” or “containing” or “including” is meant that at least the named compound, element, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if the other such compounds, material, particles, method steps have the same function as what is named. It is also noted that the term “comprising” is intended to be open and permitsbut does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of’ is thus also encompassed and disclosed.

[0265] References to “one embodiment,” “an embodiment,” “example embodiment,” “some embodiments,” “certain embodiments,” “various embodiments,” etc., indicate that the embodiment(s) of the disclosed elements or techniques so described can include a particular feature, structure, or characteristic, but not every embodiment necessarily includes the particular feature, structure, or characteristic. Further, repeated use of the phrase “in one embodiment” does not necessarily refer to the same embodiment, although it can refer to the same embodiment.

[0266] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0267] Throughout this description, various components can be identified having specific values or parameters, however, these items are provided as exemplary embodiments. Indeed, the exemplary embodiments do not limit the various aspects and concepts of the present disclosure as many comparable parameters, sizes, ranges, and / or values can be implemented. The terms “first,” “second,” and the like, “primary,” “secondary,” and the like, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

[0268] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control.

Claims

CLAIMS1. A system for adeno-associated virus (AAV) production, the system comprising:(a) a first nucleic acid molecule encoding an adenovirus L4-33K protein; and(b) a second nucleic acid molecule comprising adenovirus genes E2A, L4, E4, and VA RNA.

2. The system of claim 1, wherein the first nucleic acid molecule is present in a first vector or first adenovirus and wherein the second nucleic acid molecule is present in a second vector or second adenovirus, wherein the molar ratio of first vector or first helper adenovirus molecule to the second vector or second adenovirus is between 10:1 to 1:10, between 5: 1 and 1 :5, between 3: 1 and 1 :3, between 2:1 and 1 :2; or between 1.5:1 and 1:1.5; or wherein the first nucleic acid molecule and second nucleic acid molecule are present in a single vector or a single adenovirus.

3. The system of claim 1 or 2, wherein the first nucleic acid molecule further comprises an EF1A promoter.

4. The system of any one of claims 1-3, wherein the adenovirus genes are wild type.

5. The system of any one of claims 1-4, wherein the adenovirus genes are derived from a mutant virus.

6. The system of claim 5, wherein the mutant virus is a replication defective mutant, a life cycle defective mutant, or a temperature sensitive mutant.

7. The system of any one of claims 1-6, wherein the adenovirus (Ad) genes are serotype Ad2 genes or Ad5 genes.

8. The system of any one of claims 1-7, further comprising a third nucleic acid molecule encoding AAV replication (Rep) and AAV capsid (Cap) proteins.

9. The system of claim 8, wherein the Rep protein serotype and the Cap protein serotype are the same.

10. The system of claim 8, wherein the Rep protein serotype and the Cap protein serotype are different.

11. The system of any one of claims 8-10, wherein the third nucleic acid molecule further comprises a transgene flanked by inverted terminal repeat (ITR) sequences.

12. The system of claim 11 , wherein the first nucleic acid molecule is present in a first vector or first adenovirus and wherein the third nucleic acid molecule is present in a third vector or third adenovirus, wherein the molar ratio of first vector or first adenovirus molecule to the third vector or third adenovirus is between 10:1 to 1:10, between 5:1 and 1 :5, between 3:1 and 1:3, between 2:1 and 1:2; or between 1.5:1 and 1:1.5.

13. The system of claim 11 or 12, wherein the first nucleic acid and the second nucleic acid are present in a first vector or first adenovirus and the third nucleic acid is present in a third vector or a third adenovirus.

14. The system of any one of claims 8-10, further comprising a fourth nucleic acid molecule nucleic acid molecule encoding transgene flanked by inverted terminal repeat (ITR) sequences.

15. The system of any one of claims 1-14, wherein the second nucleic acid molecule further comprises an adenovirus pIX gene.

16. The system of any one of claims 1-15, wherein the second nucleic acid molecule does not include one or more of an adenovirus E1A gene and an adenovirus E1B gene.

17. An AAV producing cell line comprising the system of any of claims 1-16.

18. The AAV producing cell line of claim 17, wherein expression of the Adenovirus L4- 33K protein is enhanced as compared to a corresponding cell line comprising the adenovirus genes E2A, L4, E4, and VA RNA and not the first nucleic acid molecule encoding the L4-33K protein.

19. The AAV producing cell line of claim 18, wherein the cell line produces AAV with an enhanced titer in vector genomes per ml (vg / mL) as compared to the AAV titer of the corresponding cell line (vg / mL).

20. The AAV producing cell line of claim 19, wherein the enhanced titer is increased 2- fold than the AAV titer of the corresponding cell line.

21. An AAV production system comprising:(i) (a) a cell line having a genome stably integrated with one or more nucleic acid molecules encoding AAV Rep and AAV Cap proteins, an adenovirus L4-33K protein, and a transgene flanked by inverted terminal repeat (ITR) sequences; and(b) an adenovirus;(ii) (a) a cell line having a genome stably integrated with one or more nucleic acid molecules encoding AAV Rep and AAV Cap proteins, and a transgene flanked by inverted terminal repeat (ITR) sequences; and(b) an adenovirus encoding L4, E2A, E4 and VA RNA genes and a L4-33K protein; or(iii) (a) a cell line having a genome stably integrated with one or more nucleic acid molecules encoding AAV Rep and AAV Cap proteins, an adenovirus L4-33K protein, and a transgene flanked by inverted terminal repeat (ITR) sequences; and(b) an adenovirus encoding L4, E2A, E4 and VA RNA genes and a L4-33K protein.

22. The system of claim 21, wherein the adenovirus comprises L4, E2A, E4 and VA RNA genes.

23. The system of claim 21 or 22, wherein the adenovirus is wild type.

24. The system of claim 21 or 22, wherein the adenovirus is a mutant virus.

25. The system of claim 24, wherein the mutant virus is a replication defective mutant, a life cycle defective mutant, or a temperature sensitive mutant.

26. The system of any one of claims 21-25, wherein the adenovirus serotype is Ad2 or Ad5.

27. The system of any one of claims 21-26, wherein the cell line is an insect cell line or a mammalian cell line.

28. The system of any one of claims 21-27, wherein the cell line is an Sf9 cell line.

29. The system of any one of claims 21-27, wherein the cell line is a CHO cell line, Vero cell line, HeLa cell line, MDCK cell line, BHK cell line, A549 cell line, amniocyte cell line, or HEK293 cell line.

30. The system of claim 29, wherein the cell line is a HeLa S3 or HEK293 cell line.

31. The system of any one of claims 21-30, wherein the cell line expresses an Adenovirus E1A protein and / or an Adenovirus E1B protein.

32. The system of any one of claims 21-31 , wherein the produced AAV is serotype AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, a variant thereof, or a pseudo-serotype AAV.

33. A method for production of an adeno-associated virus (AAV), comprising:(a) integrating into a cell genome one or more nucleic acid molecules encoding AAV Rep and AAV Cap proteins, a transgene flanked by inverted terminal repeat (ITR) sequences; and an adenovirus L4-33K protein;(b) integrating into the cell genome adenovirus genes E2A, L4, E4, and VA RNA or Infecting the cell genome with an adenovirus;(c) expanding the cell and producing the AAV within the cell; and(d) isolating the AAV.

34. The method of claim 33, wherein the cell is an insect cell or a mammalian cell.

35. The method of claim 33 or 34, wherein the cell is an Sf9 cell.

36. The method of any one of claims 33 or 34, wherein the cell is a CHO cell, Vero cell, HeLa cell, MDCK cell, BHK cell, A549 cell, amniocyte, or HEK293 cell.

37. The method of claim 33, 34, or 36, wherein the cell is a HeLa S3 or HEK293 cell.

38. The method of any one of claims 33-37, wherein the cell expresses an AdenovirusE1A protein and / or an Adenovirus E1B protein.

39. The method of any one of claims 33-38, wherein the produced AAV is serotype AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, a variant thereof, or a pseudo-serotype AAV.

40. The method of any one of claims 33-39, whereby expression of the Adenovirus L4-33K protein is enhanced in the cell as compared to a corresponding cell comprising the adenovirus genes E2A, L4, E4, and VA RNA and not the nucleic acid molecule encoding the L4-33K protein.

41. The method of claim 40, whereby the AAV titer is enhanced in vector genomes per ml (vg / mL) as compared to the AAV titer of the corresponding cell (vg / mL).

42. The method of claim 41, wherein the enhanced AAV titer is increased by 2-fold or greater as compared to the AAV titer of the corresponding cell.

43. An adeno-associated virus (AAV) production system comprising:(a) a cell;(b) a first vector comprising at least one nucleic acid molecule encoding an adenovirus L4- 33K protein;(c) a second vector comprising one or more nucleic acid molecules encoding adenovirus genes E2A, L4, E4, and VA RNA; and(d) a third vector comprising one or more nucleic acid molecules encoding AAV Rep and AAV Cap proteins and a transgene flanked by inverted terminal repeat (ITR) sequences; or a third vector comprising one or more nucleic acid molecules encoding AAV Rep and AAV Cap proteins and a fourth vector comprising a transgene flanked by ITR sequences.

44. The system of claim 43, wherein the cell is an insect cell or a mammalian cell.

45. The system of claim 43 or 44, wherein the cell is an Sf9 cell.

46. The system of claim 43 or 44, wherein the cell is a CHO cell, Vero cell, HeLa cell,MDCK cell, BHK cell, A549 cell, amniocyte, or HEK293 cell.

47. The system of claim 43, 44 or 46, wherein the cell is a HeLa S3 or HEK293 cell.

48. The system of any one of claims 43-47, wherein the cell expresses an adenovirus E1A protein and / or an adenovirus E1 B protein.

49. A method of producing an adeno-associated virus (AAV), comprising:(a) transfecting a cell with two or more vectors comprising:(i) one or more nucleic acid molecules encoding an adenovirus L4-33K protein;(ii) one or more nucleic acid molecules encoding adenovirus genes E2A, L4, E4, and VA RNA;(iii) one or more nucleic acid molecules encoding an AAV capsid (Cap) protein and one or more nucleic acid molecules encoding an AAV replication (Rep) protein; and(iv) one more nucleic acid molecules encoding a transgene flanked by inverted terminal repeat (ITR) sequences;(b) culturing the cell under conditions suitable for producing the AAV; and(c) isolating the AAV.

50. The method of claim 49, wherein:(A) one of the two or more vectors comprises the one or more nucleic acid molecules encoding an AAV capsid (Cap) protein and one or more nucleic acid molecules encoding an AAV replication (Rep) protein; and the one more nucleic acid molecules encoding a transgene flanked by inverted terminal repeat (ITR) sequences;(B) one of the two or more vectors comprises the one or more nucleic acid molecules encoding adenovirus genes E2A, L4, E4, and VA RNA, and the one more nucleic acid molecules encoding the transgene flanked by ITR sequences;(C) one of the two or more vectors comprises the one or more nucleic acid molecules encoding an AAV capsid (Cap) protein and one or more nucleic acid molecules encoding an AAV replication (Rep) protein; and the one or more nucleic acid molecules encoding adenovirus genes E2A, L4, E4, and VA RNA;(D) wherein one of the two or more vectors comprises the one or more nucleic acid molecules encoding an AAV capsid (Cap) protein and one or more nucleic acid molecules encoding an AAV replication (Rep) protein; and the one or more nucleic acid molecules encoding the adenovirus L4-33K protein; or(E) one of the two or more vectors comprises the one more nucleic acid molecules encoding a transgene flanked by inverted terminal repeat (ITR) sequences; and the one or more nucleic acid molecules encoding the adenovirus L4-33K protein.

51. The method of claim 49, wherein the two or more vectors comprise:(a) a first vector comprising the one or more nucleic acid molecules encoding the adenovirus L4-33K protein;(b) a second vector comprising the one or more nucleic acid molecules encoding adenovirus genes E2A, L4, E4, and VA RNA; and(c) a third vector comprising the one or more nucleic acid molecules encoding the AAV Cap protein and the one or more nucleic acid molecules encoding the AAV Rep protein, and optionally the one more nucleic acid molecules encoding the transgene flanked by ITR sequences.

52. The method of claim 49, wherein the two or more vectors comprise:(a) a first vector comprising the one or more nucleic acid molecules encoding the adenovirus L4-33K protein;(b) a second vector comprising the one or more nucleic acid molecules encoding adenovirus genes E2A, L4, E4, and VA RNA;(c) a third vector comprising the one or more nucleic acid molecules encoding the AAV Cap protein and the one or more nucleic acid molecules encoding the AAV Rep protein, and(d) a fourth vector comprising the one more nucleic acid molecules encoding the transgene flanked by ITR sequences.

53. The method of claim 49, wherein the two or more vectors comprise:(a) a first vector comprising the one or more nucleic acid molecules encoding the adenovirus L4-33K protein;(b) a second vector comprising the one or more nucleic acid molecules encoding adenovirus genes E2A, L4, E4, and VA RNA, and the one more nucleic acid molecules encoding the transgene flanked by ITR sequences; and(c) a third vector comprising the one or more nucleic acid molecules encoding the AAV Cap protein and the one or more nucleic acid molecules encoding the AAV Rep protein.

54. The method of claim 49, wherein the two or more vectors comprise:(a) a first vector comprising the one or more nucleic acid molecules encoding the adenovirus L4-33K protein;(b) a second vector comprising the one or more nucleic acid molecules encoding adenovirus genes E2A, L4, E4, and VA RNA, and the one more nucleic acid molecules encoding the transgene flanked by ITR sequences; and(c) a third vector comprising the one or more nucleic acid molecules encoding the AAV Cap protein and the one or more nucleic acid molecules encoding the AAV Rep protein.

55. The method of any one of claims 49-54, wherein the two or more vectors further comprise one or more nucleic acid molecules encoding an adenovirus pIX gene.

56. The method of any one of claims 49-55, wherein the two or more vectors do not include one or more of an adenovirus E1A gene and an adenovirus E1 B gene.

57. The method of any one of claims 49-56, wherein the cell is an insect cell or a mammalian cell.

58. The method of any one of claims 49-57, wherein the cell is an Sf9 cell.

59. The method of any one of claims 49-57, wherein the cell is a CHO cell, Vero cell, HeLa cell, MDCK cell, BHK cell, A549 cell, amniocyte, or HEK293 cell.

60. The method of any one of claims 49-57 and 59, wherein the cell is a HeLa S3 or HEK293 cell.

61. The method of any one of claims 49-60, wherein the cell expresses an adenovirus E1A protein and / or an adenovirus E1 B protein.

62. The method of any one of claims 49-61 , the produced AAV is serotype AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, a variant thereof, or a pseudo-serotype AAV.

63. The method of any one of clams 49-62, whereby expression of the L4-33K protein is enhanced in the cell as compared to a corresponding cell comprising the adenovirus genes E2A, L4, E4, and VA RNA, which has not been transfected the one or more nucleic acid molecules encoding the adenovirus L4-33K protein.

64. The method of claim 63, whereby the AAV titer is enhanced in vector genomes per ml (vg / mL) as compared to the AAV titer of the corresponding cell (vg / mL).

65. The method of claim 64, wherein the enhanced AAV titer is increased by 2-fold or greater as compared to the AAV titer of the corresponding cell.