INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created Oct. 10, 2018, is named U119670031US01-SEQ-EPG.txt and is 19,249 bytes in size.

BACKGROUND

Adeno-associated virus (AAV) has emerged as one of the most promising viral vectors for human gene therapy. Recombinant adeno-associated virus (rAAV) is being evaluated in gene therapy clinical trials for hemophilia B, malignant melanoma, cystic fibrosis, and other diseases. While large scale production of rAAV using mammalian cell culture systems has been historically problematic, AAV production systems using insect cells have been shown to be a viable alternative. However, the different cellular environment has provided challenges with regard to viral particle assembly of some AAV serotypes.

SUMMARY

Provided herein are compositions and methods related to the production of rAAV in host producer cells (e.g., in insect cells). The biological potency (e.g., infectivity) of rAAV particles partly depends on the virion capsid composition, for example, the stoichiometric ratios of the capsid proteins VP1, VP2, and VP3. Some rAAV serotypes (e.g., AAVS, AAV8, and AAV9) manufactured in a heterologous system such as insect cells (e.g., Sf9 cells) are characterized by abnormal VP1 expression levels. Upon viral assembly, the abnormal VP1 expression results in the production of viral particles comprised of improper capsid protein ratios, which hinders the ability of the particles to efficiently transduce cells. Methods of obtaining useful stoichiometric ratios of capsid proteins are complicated by results indicating that an overabundance of VP1 can impair the insect cell's ability to efficiently package the viral particle while low levels of VP1 can result in particles with inefficient transduction. Thus, with respect to insect cell rAAV production systems, it remains a challenge using current techniques to produce increased amounts of infective particles without negatively impacting particle assembly.

Methods and compositions described herein provide recombinant VP1 genes comprising a modified Kozak sequence associated with the VP1 translation initiation site. Recombinant VP1 genes described in this application are useful to produce infective rAAV particles comprising gene of interest (e.g., for subsequent research and/or therapeutic uses). In some embodiments, recombinant VP1 genes described in this application can be used to produce rAAV particles of one or more serotypes in insect cells. In some embodiments, methods and compositions described in this application can be used to screen and identify recombinant VP1 genes of one or more serotypes that are useful to produce rAAV particles of one or more serotypes in insect cells and/or in other producer cells of interest (e.g., in mammalian cells). In some embodiments, one or more recombinant VP1 genes can be useful to produce stable and/or infective rAAV particles that contain recombinant genomes of interest at relatively high frequencies and/or with relatively low rates of mis-packaged nucleic acids.

In some aspects, the disclosure provides compositions and methods useful in the production of rAAV derived from an AAV5 serotype. In some aspects, the disclosure provides compositions and methods useful in the production of rAAV derived from an AAV8 serotype. In yet other aspects, the disclosure provides compositions and methods useful in the production of rAAV derived from an AAV9 serotype.

In some aspects, the disclosure provides a nucleic acid comprising a nucleotide sequence encoding a modified Kozak sequence and AAV VP1, VP2, and VP3 capsid proteins, wherein the modified Kozak sequence is selected from SEQ ID NOs: 2-11 (Table 1). In some embodiments, the modified Kozak sequence is SEQ ID NO: 8 or SEQ ID NO: 9. In some embodiments, the VP1, VP2, and VP3 capsid proteins are derived from an AAV5 serotype. In some embodiments, the VP1, VP2, and/or VP3 capsid proteins are variant AAV5 capsid proteins (e.g., they are encoded by a sequence containing one or more mutations and that encodes a capsid protein having one or more amino acid substitutions relative to a corresponding wild-type capsid protein).

In some embodiments, the nucleic acid further comprises a promoter sequence. In some embodiments, the promoter sequence is a polyhedrin (polh) promoter sequence. In some embodiments, the modified Kozak sequence contains an initiation codon for translation of the VP1 capsid protein. In some embodiments, the initiation codon for translation of the VP1 capsid protein is AUG. In some embodiments, the nucleic acid is packaged in a viral particle (e.g., a baculovirus particle). In some embodiments, the disclosure provides an insect cell comprising the nucleic acid.

In some aspects, the disclosure relates to a method of producing rAAV in an insect cell, wherein the rAAV is derived from an AAV5 serotype, the method comprising (a) transfecting an insect cell with: (i) a baculovirus comprising a nucleic acid comprising a nucleotide sequence encoding a modified Kozak sequence and AAV5 VP1, VP2, and VP3 capsid proteins, wherein the modified Kozak sequence is selected from SEQ ID NOs: 2-11, (ii) a baculovirus comprising a nucleotide sequence encoding an AAV Rep protein, (iii) a baculovirus comprising two AAV inverted terminal repeat (ITR) nucleotide sequences flanking a gene of interest operably linked to a promoter sequence; (b) culturing the insect cell under conditions suitable to produce rAAV; and (c) recovering the rAAV from the insect cell. In some embodiments, the insect cell is an Sf9 cell.

In some aspects, the disclosure provides a nucleic acid comprising a nucleotide sequence encoding a modified Kozak sequence and AAV VP1, VP2, and VP3 capsid proteins, wherein the modified Kozak sequence is selected from SEQ ID NOs: 13-32 (Table 2). In some embodiments, the modified Kozak sequence is SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32. In some embodiments, the VP1, VP2, and VP3 capsid proteins are derived from an AAV8 serotype. In some embodiments, the VP1, VP2, and/or VP3 capsid proteins are variant AAV8 capsid proteins (e.g., they are encoded by a sequence containing one or more mutations and that encodes a capsid protein having one or more amino acid substitutions relative to a corresponding wild-type capsid protein).

In some embodiments, the nucleic acid further comprises a promoter sequence. In some embodiments, the promoter sequence is a polyhedrin (polh) promoter sequence. In some embodiments, the modified Kozak sequence contains an initiation codon for translation of the VP1 capsid protein. In some embodiments, the initiation codon for translation of the VP1 capsid protein is AUG. In some embodiments, the nucleic acid is packaged in a viral particle (e.g., a baculovirus particle). In some embodiments, the disclosure provides an insect cell comprising the nucleic acid.

In some aspects, the disclosure relates to a method of producing rAAV in an insect cell, wherein the rAAV is derived from an AAV8 serotype, the method comprising (a) transfecting an insect cell with: (i) a baculovirus comprising a nucleic acid comprising a nucleotide sequence encoding a modified Kozak sequence and AAV8 VP1, VP2, and VP3 capsid proteins, wherein the modified Kozak sequence is selected from SEQ ID NO: 13-32, (ii) a baculovirus comprising a nucleotide sequence encoding an AAV Rep protein, (iii) a baculovirus comprising two AAV ITR nucleotide sequences flanking a gene of interest operably linked to a promoter sequence; (b) culturing the insect cell under conditions suitable to produce rAAV; and (c) recovering the rAAV from the insect cell. In some embodiments, the insect cell is an Sf9 cell.

In some aspects, the disclosure provides a nucleic acid comprising a nucleotide sequence encoding a modified Kozak sequence and AAV VP1, VP2, and VP3 capsid proteins, wherein the modified Kozak sequence is selected from SEQ ID NO: 34-45 (Table 3). In some embodiments, the modified Kozak sequence is SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 42. In some embodiments, the VP1, VP2, and VP3 capsid proteins are derived from an AAV9 serotype. In some embodiments, the VP1, VP2, and/or VP3 capsid proteins are variant AAV9 capsid proteins (e.g., they are encoded by a sequence containing one or more mutations and that encodes a capsid protein having one or more amino acid substitutions relative to a corresponding wild-type capsid protein).

In some embodiments, the nucleic acid further comprises a promoter sequence. In some embodiments, the promoter sequence is a polyhedrin (polh) promoter sequence. In some embodiments, the modified Kozak sequence contains an initiation codon for translation of the VP1 capsid protein. In some embodiments, the initiation codon for translation of the VP1 capsid protein is AUG. In some embodiments, the nucleic acid is packaged in a viral particle (e.g., a baculovirus particle). In some embodiments, the disclosure provides an insect cell comprising the nucleic acid.

In some aspects, the disclosure relates to a method of producing rAAV in an insect cell, wherein the rAAV is derived from an AAV9 serotype, the method comprising (a) transfecting an insect cell with: (i) a baculovirus comprising a nucleic acid comprising a nucleotide sequence encoding a modified Kozak sequence and AAV9 VP1, VP2, and VP3 capsid proteins, wherein the modified Kozak sequence is selected from SEQ ID NO: 34-45, (ii) a baculovirus comprising a nucleotide sequence encoding an AAV Rep protein, (iii) a baculovirus comprising two AAV ITR nucleotide sequences flanking a gene of interest operably linked to a promoter sequence; (b) culturing the insect cell under conditions suitable to produce rAAV; and (c) recovering the rAAV from the insect cell. In some embodiments, the insect cell is an Sf9 cell.

In some aspects, this application provides a library of nucleic acids comprising a nucleotide sequence encoding a modified Kozak sequence comprising the initiation codon for translation of an AAV VP1 capsid protein and nucleotide sequence variations in 1-6 nucleotides immediately upstream of the VP1 translation initiation codon and/or nucleotide sequence variations in 1-2 nucleotides immediately downstream of the VP1 translation initiation codon. In some embodiments, the nucleic acid comprises XXXXXX(ATG), wherein (ATG) is the VP1 initiation codon. In some embodiments, the nucleic acid comprises XXXXXX(AUG), wherein (AUG) is the VP1 initiation codon. In some embodiments, X represents any nucleotide, e.g., any nucleotide that is different from a naturally occurring nucleotide at that position in a wild-type VP1 gene for an AAV serotype of interest.

These and other aspects are described in more detail in the following description and accompanying examples and figures.

