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Definitions

• the present invention is directed to recombinantly-modified adeno-associated virus (rAAV) having improved packaging efficiency, pharmaceutical compositions comprising such rAAV, and methods for their production and use.
• the present invention is particularly directed to recombinantly-modified adeno-associated virus (rAAV) that have been further modified to comprise Cis-Elements, including replication origins, promoters and enhancers, that are capable of regulating the replication of an rAAV genome and that improve rAAV replication.
• Cis-Elements are provided within domains of the rAAV that precede and/or follow the 5’ and/or 3’ inverted terminal repeated sequences (ITR) of an rAAV.
• the invention particularly concerns the presence and the use of polynucleotide Cis-Elements that comprise actual or potential G-Quadruplex Sequences, polynucleotide Cis-Elements that comprise DNA sequences from wild-type AAV (wt AAV) and polynucleotide Cis- Elements that comprise DNA sequences from other viral genomes or from the human genome.
• polynucleotide Cis-Elements that comprise actual or potential G-Quadruplex Sequences
• polynucleotide Cis-Elements that comprise DNA sequences from wild-type AAV (wt AAV) and polynucleotide Cis- Elements that comprise DNA sequences from other viral genomes or from the human genome.
• AAV Adeno-Associated Virus
• Adeno-Associated Virus is a small, naturally-occurring, non- pathogenic virus belonging to the Dependovirus genus of the Parvoviridae (Balakrishnan, B. et al. (2014)“ Basic Biology of Adeno-Associated Virus (AAV) Vectors Used in Gene Therapy ,” Curr. Gene Ther. 14(2):86-100; Zinn, E. et al. (2014) “ Adeno-Associated Virus: Fit To Serve,” Curr. Opin. Virol. 0:90-97). Despite not causing disease, AAV is known to be able to infect humans and other primates and is prevalent in human populations (Johnson, F.B. et al.
• AAV infect a broad range of different cell types (e.g, cells of the central nervous system, heart, kidney, liver, lung, pancreas, retinal pigment epithelium or photoreceptor cells, or skeletal muscle cells). Twelve serotypes of the virus (e.g, AAV2, AAV5, AAV6, etc.), exhibiting different tissue infection capabilities (“tropisms”), have been identified (Colella, P. et al.
• AAV is a single-stranded DNA virus that is composed of approximately 4,700 nucleotides.
• the viral genome may be described as having a 5’ half and a 3’ half which together comprise the genes that encode the virus’ proteins ( Figure 1).
• the 5’ half of the AAV genome comprises the AAV rep gene, which, through the use of multiple reading frames, staggered initiating promoters (p5, pl9 and p40) and alternate splicing, encodes four non- structural Rep proteins (Rep40, Rep52, Rep68 and Rep78) that are required for viral transcription control and replication and for the packaging of viral genomes into the viral capsule (Lackner, D.F. et al.
• the 3’ half the AAV genome comprises the AAV capsid gene (cap), which encodes three capsid proteins (VP): VP1, VP2 and VP3.
• the three capsid proteins are translated from a single mRNA transcript that is controlled by a single promoter (p40 in case of AAV2).
• the 3’ half of the AAV genome also comprises the AAP gene, which encodes the AAV assembly-activating protein (AAP).
• VP monomers comprising approximately 5 copies of VP1, 5 copies of VP2, and 50 copies of VP3 self-assemble around the AAV genome to form the icosahedral protein shell (capsid) of the mature viral particle (Biining, H. et al. (2019)“Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors f Mol. Ther. Meth. Clin. Devel. 12:P248-P265; Van Vliet K.M. et al. (2008) The Role of the Adeno-Associated Virus Capsid in Gene Transfer. In: DRUG DELIVERY SYSTEMS, Jain, K.K. (eds.), Meth.
• the AAV AAP protein is believed to be required for stabilizing and transporting newly produced VP proteins from the cytoplasm into the cell nucleus.
• the 3’ half of the AAV genome also comprises the AAV X gene, which is believed to encode a protein that supports genome replication (Colella, P. etal. (2016) “Emerging Issues in AAV-Mediated In Vivo Gene Therapy f Molec. Ther. Meth. Clin. Develop. 8:87-104; Biining, H. et al. (2019)“Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors f Mol. Ther. Meth. Clin. Devel.
• AAV gene-coding sequences are flanked by two AAV-specific palindromic inverted terminal repeated sequences (ITR) of 145 nucleotides (Balakrishnan, B. et al. (2014)“ Basic Biology of AdenoAssociated Virus (AAV) Vectors Used in Gene Therapyf Curr. Gene Ther. 14(2):86-100; Colella, P. et al. (2016)“Emerging Issues in AAV-Mediated In Vivo Gene Therapyf Molec. Ther. Meth. Clin. Develop. 8:87-104).
• ITR AAV-specific palindromic inverted terminal repeated sequences
• AAV is an inherently defective virus, lacking the capacity to perform at least two critical functions: the ability to initiate the synthesis of viral-specific products and the ability to assemble such products to form the icosahedral protein shell (capsid) of the mature infectious viral particle. It thus requires a co-infecting“helper” virus, such as adenovirus (Ad), herpes simplex virus (HSV), cytomegalovirus (CMV), vaccinia virus or human papillomavirus to provide the viral-associated ( A) RNA that is not encoded by the genes of the AAV genome. Such VA RNA is not translated, but plays a role in regulating the translation of other viral genes.
• the AAV genome does not include genes that encode the viral proteins El a, Elb, E2a, and E4 of Ad; thus, these proteins must also be provided by a co-infecting“helper” virus.
• the El a protein greatly stimulate viral gene transcription during the productive infection.
• the Elb protein block apoptosis in adenovirus-infected cells, and thus allow productive infection to proceed.
• the E2a protein plays a role in the elongation phase of viral strand displacement replication by unwinding the template and enhancing the initiation of transcription.
• the E4 protein has been shown to affect transgene persistence, vector toxicity and immunogenicity (see, Grieger, J.C. et al.
• AAV viruses infect both dividing and non-dividing cells, and persist as circular episomal molecules or can be integrated into the DNA of a host cell at specific chromosomic loci (Adeno-Associated Virus Integration Sites or AAVS) (Duan, D. (2016)“ Systemic Delivery Of Adeno-Associated Viral Vectors ,” Curr. Opin. Virol. 21 : 16-25; Grieger, J.C. et al. (2012) “ Adeno-Associated Virus Vectorology, Manufacturing, and Clinical Applications ,” Meth. Enzymol. 507:229-254). AAV remains latent in such infected cells unless a helper virus is present to provide the functions needed for AAV replication and maturation.
• rAAV are typically produced using circular plasmids (“rAAV plasmid vector”).
• the AAV rep and cap genes are typically deleted from such constructs and replaced with a promoter, a b-globin intron, a cloning site into which a therapeutic gene of choice (transgene) has been inserted, and a poly-adenylation (“poly A”) site.
• the inverted terminal repeated sequences (ITR) of the rAAV are, however, retained, so that the transgene expression cassette of the rAAV plasmid vector is flanked by AAV ITR sequences (Colella, P. et al. (2016)“Emerging Issues in AAV-Mediated In Vivo Gene Therapy ,” Molec. Ther.
• the rAAV comprises a 5’ ITR, the transgene expression cassette of the rAAV, and a 3’ ITR.
• rAAV have been used to deliver a transgene to patients suffering from any of a multitude of genetic diseases (e.g, hereditary lipoprotein lipase deficiency (LPLD), Leber’s congenital amaurosis (LCA), aromatic L-amino acid decarboxylase deficiency (AADC), choroideremia and hemophilia), and have utility in new clinical modalities, such as in interfering RNA (RNAi) therapy and gene-modifying strategies such as Crispr/Cas9 (US Patents No. 8,697,359, 10,000,772, 10,113,167, 10,227,611; Lino, C.A. etal.
• LPLD hereditary lipoprotein lipase deficiency
• LCA congenital amaurosis
• AADC aromatic L-amino acid decarboxylase deficiency
• choroideremia choroideremia and hemophilia
• RNAi interfering RNA
• AAV serotype for such recombinantly-modified AAV is AAV2, which is capable of infecting cells of the central nervous system, kidney, retinal pigment epithelium and photoreceptor cells.
• AAV serotype is AAV9, which infects muscle cells, also has been widely used (Duan, D. (2016)“Systemic Delivery Of Adeno-Associated Viral Vectors ,” Curr. Opin. Virol. 21 :16-25).
• AAV serotypes are described in US Patents No.
• rAAV containing a desired transgene expression cassette are typically produced by human cells (such as HEK293) grown in suspension. Since, as described above, rAAV are defective viruses, additional functions must be provided in order to replicate and package rAAV.
• rAAV are produced by transiently transfecting cells with an rAAV plasmid vector and a second plasmid vector that comprises an AAV helper function providing polynucleotide that provides the Rep52 and Rep78 genes that are required for vector transcription control and replication, and for the packaging of viral genomes into the viral capsule (Rep40 and Rep68 are not required for rAAV production) and the cap genes that were excised from the AAV in order to produce the rAAV.
• AAV helper function providing polynucleotide that provides the Rep52 and Rep78 genes that are required for vector transcription control and replication, and for the packaging of viral genomes into the viral capsule (Rep40 and Rep68 are not required for rAAV production) and the cap genes that were excised from the AAV in order to produce the rAAV.
• the second plasmid vector may additionally comprise a non-AAV helper function providing polynucleotide that encodes the viral transcription and translation factors (Ela, Elb, E2a, VA and E4) required for AAV proliferation, so as to comprise, in concert with the rAAV, a double plasmid transfection system (Grimm, D. et al. (1998) “Novel Tools For Production And Purification Of Recombinant Adeno- Associated Virus Vectors ,” Hum. Gene Ther. 9:2745-2760; Penaud-Budloo, M. et al. (2016)“ Pharmacology of Recombinant Adeno-associated Virus Production,” Molec. Ther. Meth. Clin. Develop. 8: 166-180).
• a non-AAV helper function providing polynucleotide that encodes the viral transcription and translation factors (Ela, Elb, E2a, VA and E4) required for AAV proliferation, so as to comprise, in concert with the rAAV,
• Else of the triple plasmid transfection system has the advantage of permitting one to easily switch one cap gene for another, thereby facilitating changes in the rAAV’s serotype.
• helper plasmids rather than helper viruses, permits rAAV to be produced without additionally producing particles of the helper virus (Franqois, A. et al. (2016)“ Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls ,” Molec. Ther. Meth. Clin. Develop. 10:223-236; Matsushita, T. et al. (1998) “ Adeno-Associated Virus Vectors Can Be Efficiently Produced Without Helper Virus,” Gene Ther. 5:938-945).
• rAAV may alternatively be produced in insect cells (e.g. , sf9 cells) using baculoviral vectors (see, e.g. , US Patents No.: 9,879,282; 9,879,279; 8,945,918; 8,163,543; 7,271,002 and 6,723,551), or in HSV-infected baby hamster kidney (BHK) cells (e.g, BHK21 (Franqois, A. et al. (2016)“ Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls ,” Molec. Ther. Meth. Clin. Develop. 10:223-236).
• insect cells e.g. , sf9 cells
• BHK HSV-infected baby hamster kidney
• rAAV are typically collected and purified by one or more overnight CsCl gradient centrifugations (Zolotukhin, S. et al. (1999)“ Recombinant Adeno-Associated Virus Purification Using Novel Methods Improves Infectious Titer And Yield,” Gene Ther. 6:973-985), followed by desalting to form a purified rAAV production stock. Titers of 10 12 -10 13 infectious rAAV capsids/mL are obtainable.
• TCID50 median tissue culture infective dose
• a HeLa-derived AAV2 rep- and c «/;-expressing cell line is grown in a 96- well plate and infected with replicate 10-fold serial dilutions of the rAAV preparation, in the presence of adenovirus of serotype 5. After infection, vector genome replication is determined by quantitative PCR (qPCR) (Zen, Z. et al.
• infectious titer of an rAAV preparation can be measured using the infectious center assay (ICA).
• ICA infectious center assay
• This assay uses HeLa rep-cap cells and Ad, but, after incubation, involves transferring the cells to a membrane.
• a labeled probe that is complementary to a portion of the employed transgene is used to detect infectious centers (representing individual infected cells) via hybridization.
• G-Quadruplex Structures are formed from the stacking of three planar“G-tetrad” (also known as“guanine quartet”) structures. Each G-tetrad is formed through Hoogsteen base pairing via hydrogen bond interactions involving four deoxyguanosine residues.
• the planar structure of the G-tetrad may be stabilized by cations ( e.g ., Na + ).
• the guanines are attached to their respective polynucleotide chain(s) via“R”.
• the stacking of G-tetrad structures to form a G-Quadruplex Structure is accomplished by the spontaneous interaction and/or looping of domains of one, two or four polynucleotide chains that together comprise a G-Quadruplex Sequence ( Figures 3A-3D).
• Viral G-Quadruplex Structures have been proposed to function as steric blocks to DNA replication and transcription (Satkunanathan, S. et al. (2017)“ The Function Of DNA Binding Protein Nucleophosmin In AAV Replication f Virol. 510:46-54).
• NHE IIIi Nuclease hypersensitive element III
• a major regulator of c-MYC transcription causes that gene to be expressed at a lower level than that of a mutated NHE IIIi gene (Siddiqui-Jain, A. et al.
• Nucleophosmin is a nucleolar protein that plays a role in many diverse functions, such as genome stability, DNA duplication and transcriptional regulation through its ability to bind to single-stranded nucleic acids.
• Nucleophosmin has been reported to enhance AAV infection by acting as a chaperone protein to mobilize AAV capsids into and out of the nucleolus (Nash, K. et al. (2009) “ Identification Of Cellular Proteins That Interact With The Adeno-Associated Virus Rep Proteinf J. Virol. 83(l):454-469; Ni, T.H. et al.
• Nucleophosmin has, however, also been found to negatively regulate DNA replication by binding to G-Quadruplex Sequences (Gallo, A. et al. (2012) “ Structure of Nucleophosmin DNA-binding Domain and Analysis of Its Complex with a G- Quadruplex Sequence from the c-MYC Promoter ,” J. Biol. Chem.
• the present invention is directed to improved methods for increasing the efficiency of AAV and rAAV packaging through regulation of the replication of rAAV genomes.
• the present invention is directed to recombinantly-modified adeno-associated virus (rAAV) having improved packaging efficiency, pharmaceutical compositions comprising such rAAV, and methods for their production and use.
• the present invention is particularly directed to recombinantly-modified adeno-associated virus (rAAV) that have been further modified to comprise Cis-Elements, including replication origins, promoters and enhancers, that are capable of regulating the replication of an rAAV genome and that improve rAAV replication.
• Cis-Elements are provided within domains of the rAAV that precede and/or follow the 5’ and/or 3’ inverted terminal repeated sequences (ITR) of an rAAV.
• the invention particularly concerns the presence and the use of polynucleotide Cis-Elements that comprise actual or potential G-Quadruplex Sequences, polynucleotide Cis-Elements that comprise DNA sequences from wild-type AAV (wt AAV) and polynucleotide Cis- Elements that comprise DNA sequences from other viral genomes or from the human genome.
• rAAV recombinantly-modified adeno-associated virus
• the PI Domain is 5’ to a 5’ ITR of the rAAV;
• Cis-Element causes rAAV-producing cells to produce the rAAV at a higher production titer than would be attained with such rAAV if lacking the Cis-Element.
• the invention also provides a pharmaceutical composition that comprises:
• rAAV recombinantly-modified adeno-associated virus
• the PI Domain is 5’ to a 5’ ITR of the rAAV;
• the invention also provides a method for increasing the production titer of recombinantly-modified adeno-associated virus (rAAV), wherein the method comprises:
• the PI Domain is 5’ to a 5’ ITR of the rAAV;
• the P2 Domain is 3’ to the 5’ ITR of the rAAV and 5’ to a transgene cassette of the rAAV;
• the P3 Domain is 3’ to the transgene cassette of the rAAV and 5’ to a 3’ ITR of the rAAV;
• the invention also provides the embodiment of such recombinantly-modified adeno-associated virus (rAAV), pharmaceutical composition, or method, wherein the employed rAAV has been modified to comprise an added Cis-Element in its PI
• the invention also provides the embodiment of such recombinantly-modified adeno-associated viruses (rAAV), pharmaceutical compositions, or methods, wherein the employed rAAV has been modified to comprise an added Cis-Element in its P2
• the invention also provides the embodiment of such recombinantly-modified adeno-associated viruses (rAAV), pharmaceutical compositions, or methods, wherein the employed rAAV has been modified to comprise an added Cis-Element in its P3
• the invention also provides the embodiment of such recombinantly-modified adeno-associated viruses (rAAV), pharmaceutical compositions, or methods, wherein the employed rAAV has been modified to comprise an added Cis-Element in its P4
• the invention also provides the embodiment of such recombinantly-modified adeno-associated viruses (rAAV), pharmaceutical compositions, or methods, wherein the employed rAAV has been modified to comprise an added Cis-Element in its PI Domain and in one or more of its P2, P3 or P4 Domain.
• rAAV recombinantly-modified adeno-associated viruses
• the invention also provides the embodiment of such recombinantly-modified adeno-associated viruses (rAAV), pharmaceutical compositions, or methods, wherein the employed rAAV has been modified to comprise an added Cis-Element in its P2 Domain and in one or more of its P3 or P4 Domain.
• rAAV recombinantly-modified adeno-associated viruses
• the invention also provides the embodiment of such recombinantly-modified adeno-associated viruses (rAAV), pharmaceutical compositions, or methods, wherein the employed rAAV has been modified to comprise an added Cis-Element in its P3
• the invention also provides the embodiment of such recombinantly-modified adeno-associated viruses (rAAV), pharmaceutical compositions, or methods, wherein the added Cis-Element forms a G-Quadruplex Structure in the employed rAAV.
• rAAV recombinantly-modified adeno-associated viruses
• the invention also provides the embodiment of such recombinantly-modified adeno-associated viruses (rAAV), pharmaceutical compositions, or methods, wherein an added Cis-Element is selected from the group consisting of:
• the invention also provides the embodiment of such recombinantly-modified adeno-associated viruses (rAAV), pharmaceutical compositions, or methods, wherein the transgene cassette encodes a protein, or comprises a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition.
• the invention also provides the embodiment of such recombinantly-modified adeno-associated viruses (rAAV), pharmaceutical compositions, or methods, wherein the employed rAAV belongs to the rAAVl, rAAV2, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9 or rAAVl 0 serotype, or to a hybrid of the serotypes.
• the invention also provides the embodiment of such recombinantly-modified adeno-associated viruses (rAAV), pharmaceutical compositions, or methods, wherein the employed rAAV belongs to the rAAV2, rAAV5, or rAAV9 serotype, or to a hybrid of the serotypes.
• rAAV recombinantly-modified adeno-associated viruses
• the invention also provides the embodiment of such recombinantly-modified adeno-associated viruses (rAAV), pharmaceutical compositions, or methods, wherein the cells are human embryonic kidney cells, baby hamster kidney cells or sf9 insect cells.
• rAAV recombinantly-modified adeno-associated viruses
• the invention also provides the embodiment of such recombinantly-modified adeno-associated viruses (rAAV), pharmaceutical compositions, or methods, wherein the cells are HEK293 human embryonic kidney cells.
• rAAV recombinantly-modified adeno-associated viruses
• the invention also provides the embodiment of such recombinantly-modified adeno-associated viruses (rAAV), pharmaceutical compositions, or methods, wherein the cells are BHK21 baby hamster kidney cells.
• rAAV recombinantly-modified adeno-associated viruses
• the invention also provides such recombinantly-modified adeno-associated viruses (rAAV) and pharmaceutical compositions, wherein the transgene cassette encodes a protein, or comprises a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition, for use in the treatment of the genetic or heritable disease or condition.
• rAAV recombinantly-modified adeno-associated viruses
• Figure 1 provides a schematic genetic map of the wild-type (Wt) AAV genome.
• FIG. 2 provides a schematic of the structural domain of the wild-type AAV2 genome (1 ), a recombinant AAV (rAAV) (2), complementing“AAV helper plasmid” (3) and an adenovirus helper plasmid (“Ad helper plasmid”) (4).
• the wild-type (Wt) AAV2 (1 ) is composed of AAV-specific palindromic inverted terminal repeated sequences (ITR), a 5’ half containing genes that encode the Rep proteins and a 3’ half containing genes that encode the Cap proteins.
• the rAAV (2) is formed by replacing the Rep- and Cap-encoding genes of the wild-type (Wt) AAV2 (1 ) with a transgene cassette that comprises a promoter (Pro), the exogenous transgene of interest, and a polyadenylation site (pA).
• the complementing AAV helper plasmid (3) provides Rep and Cap proteins.
• the Ad helper plasmid (4) provides adenovirus proteins Ela, Elb, E2a, VA and E4.
• Figures 3A-3D show illustrative G-Quadruplex Structures formed by the stacking of multiple G-tetrads.
• Figures 3A-3D depict such G-tetrad as gray planar rectangles whose vertices are connected to the polynucleotide backbone.
• the G- Quadruplex Structures may be formed from a single polynucleotide chain ( Figures 3A- 3B), that spontaneously loops back upon itself, from two polynucleotide strands ( Figure 3C) that loop back upon each other, or from four polynucleotide chains (Figure 3D) that loop back upon each other.
• polypeptide chains may form loops in either an anti parallel manner (e.g ., Figure 3A) or in a parallel manner (e.g ., Figure 3B) (see, Harris, L.M. et al. (2015)“ G-Quadruplexes In Pathogens: A Common Route To Virulence ControlT PLoS Pathog. 1 l(2):el004562 (pages 1-15).
• Figure 4 shows a map of the AAV helper plasmid vector pAAV-RC2.
• Figure 5 shows a map of the non-AAV helper plasmid vector pHelper-Kan.
• Figure 6 shows a map of the rAAV plasmid vector pAV-CMV-EGFP.
• Figure 7 shows a map of the rAAV plasmid vector pAV-TBG-EGFP.
• Figure 8 shows the overall structure and approach followed for the development of the exemplary rAAV constructs described herein (ITR: adeno- associated virus (AAV)-specific palindromic inverted terminal repeated sequences; Pro: promoter. Gene: transgene; PolyA: polynucleotide comprising poly- deoxyadenosine sequence; CisE: polynucleotide comprising a Cis-Element).
• ITR adeno- associated virus (AAV)-specific palindromic inverted terminal repeated sequences
• Pro promoter.
• Gene transgene
• PolyA polynucleotide comprising poly- deoxyadenosine sequence
• CisE polynucleotide comprising a Cis-Element
• Figures 9A-9B show the increase in rAAV production titers obtained by introducing a Cis-Element within the P2 Domain of the rAAV plasmid vector pAV- TBG-EGFP.
• Figure 9A shows the P2 Domain of the rAAV plasmid vector pAV- TBG-EGFP.
• Figure 9B shows the production titers of rAAV obtained using rAAV plasmid vectors containing any of Cis-Elements CisEl-CisE27 (Table 1), relative to that obtained using the parental rAAV plasmid vector, pAV-TBG-EGFP.
• the production titers of rAAV were obtained using the parental or derivative rAAV plasmids in a triple plasmid transfection system with a helper plasmid providing the AAV rep and cap functions and an Ad helper plasmid that provided the required adenoviral functions.
• Figures 10A-10B show the increase in rAAV production titers obtained by introducing a Cis-Element within the PI Domain of the rAAV plasmid vector pAV- TBG-EGFP ( Figure 10A).
• Figure 10B shows the production titers of rAAV obtained using rAAV plasmid vectors containing any of Cis-Elements CisEl, CisE20, CisE21, CisE27, CisE28, CisE29, or CisE30 (Table 1), relative to that obtained using the parental rAAV plasmid vector, pAV-TBG-EGFP.
• the production titers of rAAV were obtained using the parental or derivative rAAV plasmid vectors in a triple plasmid transfection system with an AAV helper plasmid providing the AAV rep and cap functions and an Ad helper plasmid that provided the required adenoviral functions.
• Figures 11A-11B show the increase in rAAV production titers obtained by introducing the same Cis-Element within the PI Domain or P2 Domain of the rAAV plasmid vector pAV-TBG-EGFP ( Figure 11 A).
• Figure 11B shows the production titers of rAAV obtained using rAAV plasmids containing any of Cis-Elements CisEl, CisE20, or CisE21 (Table 1), relative to that obtained using the parental rAAV plasmid, pAV-TBG-EGFP.
• FIGS. 12A-12B show the effect of Cis-Element orientation on the ability of a Cis-Element, inserted within the PI Domain the rAAV plasmid vector pAV-TBG- EGFP, to cause an increase in rAAV production titer ( Figure 12A).
• Figure 12B shows the production titers of rAAV obtained using rAAV plasmid vectors containing Cis- Element CisE21 (Table 1) in the forward orientation (SEQ ID NO:41) (“CisE21-For- Pl”) or in the reverse orientation (SEQ ID NO:42) (“CisE21-Rev-Pl”), relative to that obtained using the parental rAAV plasmid vector, pAV-TBG-EGFP.
• the production titers of rAAV were obtained using the parental or derivative rAAV plasmids in a triple plasmid transfection system with an AAV helper plasmid providing the AAV rep and cap functions and an Ad helper plasmid that provided the required adenoviral functions.
• Figures 13A-13D show the effect of Cis-Element orientation on the ability of a Cis-Element, inserted within both the PI Domain the rAAV plasmid vector pAV- TBG-EGFP and the P4 Domain of such plasmid vector to cause an increase in rAAV production titer ( Figure 13A).
• Figure 13B shows the production titers of rAAV obtained using rAAV plasmid vectors containing different Cis-Elements (Cis-Element CisE21, CisE22, CisE23, CisE24, CisE25, CisE26, CisE28, CisE29, CisE31, CisE32, CisE33, CisE34, or CisE35) within the PI Domain, while maintaining the same Cis- Element (CisE30-Rev) within the P4 Domain of the rAAV plasmid vector.
• Cis-Element CisE21, CisE22, CisE23, CisE24, CisE25, CisE26, CisE28, CisE29, CisE31, CisE32, CisE33, CisE34, or CisE35 within the PI Domain, while maintaining the same Cis- Element (CisE30-Rev) within the P4 Domain of the r
• Figure 13C shows the production titers of rAAV obtained using rAAV plasmid vectors containing different Cis-Elements (Cis-Element CisE21, CisE22, CisE23, CisE24, CisE25, CisE27, CisE28, CisE32, CisE33, or CisE34) within its PI Domain and Cis- Element CisE35-Rev within its P4 Domain.
• Cis-Element CisE21, CisE22, CisE23, CisE24, CisE25, CisE27, CisE28, CisE32, CisE33, or CisE34 within its PI Domain and Cis- Element CisE35-Rev within its P4 Domain.
• Figure 13D shows the production titers of rAAV obtained using rAAV plasmid vectors containing different Cis-Elements Cis- Element CisE22-Rev, CisE27-Rev, CisE29-Rev, or CisE35-Rev) within the P4 Domain, while maintaining the same Cis-Element (CisE28) within the PI Domain of the rAAV plasmid vector.
• the production titers of rAAV were obtained using the parental or derivative rAAV plasmids in a triple plasmid transfection system with an AAV helper plasmid providing the AAV rep and cap functions and an Ad helper plasmid that provided the required adenoviral functions.
• the present invention is directed to recombinantly-modified adeno-associated virus (rAAV) having improved packaging efficiency, pharmaceutical compositions comprising such rAAV, and methods for their production and use.
• the present invention is particularly directed to recombinantly-modified adeno-associated virus (rAAV) that have been further modified to comprise Cis-Elements, including replication origins, promoters and enhancers, that are capable of regulating the replication of an rAAV genome and that improve rAAV replication.
• Cis-Elements are provided within domains of the rAAV that precede and/or follow the 5’ and/or 3’ inverted terminal repeated sequences (ITR) of an rAAV.
• the invention particularly concerns the presence and the use of polynucleotide Cis-Elements that comprise actual or potential G-Quadruplex Sequences, polynucleotide Cis-Elements that comprise DNA sequences from wild-type AAV (wt AAV) and polynucleotide Cis- Elements that comprise DNA sequences from other viral genomes or from the human genome.
• polynucleotide Cis-Elements that comprise actual or potential G-Quadruplex Sequences
• polynucleotide Cis-Elements that comprise DNA sequences from wild-type AAV (wt AAV) and polynucleotide Cis- Elements that comprise DNA sequences from other viral genomes or from the human genome.
• the present invention is based in part on the recognition that high levels of DNA replication increase both the amount of rAAV genomes particles and, consequently, the efficiency of rAAV packaging, and thus result in high production titers of rAAV stocks.
• Such desired high levels of DNA replication can be attained by modifying rAAV or rAAV plasmid vectors to contain additional polynucleotides that comprise replication origins, promoters, enhancers, etc.
• Cis-Elements Because such polynucleotides act to increase the replication of rAAV vectors on which they are present, they are referred to herein as“Cis-Elements.”
• the invention encompasses recombinant AAV vectors and rAAV plasmid vectors that carry such Cis-Elements and their use in the production of novel stable cell lines capable of generating high titer rAAV preparations.
• the Cis-Elements of the present invention are preferably introduced into an rAAV plasmid vector. Such introduction is preferably accomplished using well-known methods of recombinant DNA technology.
• the term“AAV” is intended to denote adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms.
• the term “rAAV” is intended to denote a recombinantly-modified version of AAV that comprises a polynucleotide sequence not of AAV origin ( i.e ., a polynucleotide heterologous to AAV).
• the rAAV may be single-stranded or double-stranded, and may be composed of deoxyribonucleotides or ribonucleotides.
• AAV helper functions denotes AAV proteins (e.g. , Rep and Cap) and/or polynucleotides of AAV that are required for the replication and packaging of an rAAV.
• AAV helper functions are provided by an“AAV helper function-providing polynucleotide,” which as such term is used herein is a virus, plasmid vector, a non-plasmid vector, or a polynucleotide that has been integrated into a cellular chromosome, that provides AAV helper functions.
• AAV helper plasmids that may be used in accordance with the present invention to provide AAV helper functions, such as pAAV-RC (Agilent; Addgene; Cell Biolabs), pAAV-RC2 (Cell Biolabs), etc., are commercially available.
• Plasmid pAAV-RC2 (SEQ ID NO:l; Figure 4) is an AAV helper plasmid that may be used in accordance with the present invention to provide AAV helper functions.
• residues 85-1950 of pAAV-RC2 encode the Rep protein, Rep78 (with residues 484-663 corresponding to the P19 promoter, residues 1464-1643 corresponding to the P40 promoter and residues 1668-1676 being a donor site); residues 1967-4174 encode the capsid protein, VP1; residues 1992-2016 encodes a portion of the Rep68 protein; residues 4175-4256 encode a polyA sequence; residues 4610-4626 are M13 Rev sequences; residues 4634-4650 are Lac operator sequences; 4658-4688 are Lac promoter sequences; residues 4951-5675 correspond to pMB ori sequences, residues 5771-6631 encode an ampicillin resistance determinant; and residues 6632- 6730 are bla promoter sequences ( Figure 4).
• non-AAV helper functions denotes proteins of Ad, CMV, HSV or other non-AAD viruses (e.g, El a, Elb, E2a, VA and E4) and/or polynucleotides of Ad, CMV, HSV or other non-AAD viruses that are required for the replication and packaging of an rAAV.
• non-AAV helper functions are provided by a“non-AAV helper function-providing polynucleotide,” which as such term is used herein is a virus, plasmid vector, a non-plasmid vector, or a polynucleotide that has been integrated into a cellular chromosome, that provides non-AAV helper functions.
• the vector, pHelper and derivatives thereof are suitable non-AAV helper function providing polynucleotide (see, e.g., Matsushita, T. et al.
• Plasmid pHelper-Kan (SEQ ID NO:2; Figure 5) is a non-AAV helper function-providing polynucleotide that may be used in accordance with the present invention to provide non-AAV helper functions.
• AAV helper function-providing polynucleotides and non-AAV helper function-providing polynucleotides are typically employed in concert with an rAAV plasmid vector to comprise a triple plasmid transfection system.
• rAAV plasmid vectors e.g ., pAV-CMV-EGFP, pGOI, etc. (Cell Biolabs, Inc., Invitrogen and Stratagene) may be used in accordance with the present invention.
• An illustrative rAAV plasmid vector that may be used in accordance with the present invention is pAV-CMV-EGFP (SEQ ID NO:3; Figure 6) which comprises a 5’ ITR, a U6 promoter, CMV enhancer and promoter sequences, a polynucleotide encoding the enhanced green fluorescent protein (EGFP) (Gambotto, A. el al. (2000)“ Immunogenicity Of Enhanced Green Fluorescent Protein (EGFP) In BALB/C Mice: Identification Of An H2-Kd-Restricted CTL Epitope ,” Gene Ther. 7(23):2036-2040; Tsien, R.Y. (1998)“The Green Fluorescent Protein ,” Annu. Rev.
• EGFP enhanced green fluorescent protein
• residues 1-128 of pAV-CMV-EGFP correspond to the 5’ ITR; residues 201-441 are U6 promoter sequences; residues 562-865 are human cytomegalovirus (CMV) immediate early enhancer sequences; residues 866-1068 comprise the CMV immediate early promoter; residues 1192-1911 comprise a mammalian codon-optimized polynucleotide that encodes the EGFP; residues 1918- 1941 encode the FLAG-tag; residues 1951-1968 encode the 6xHis-tag; residues 2139- 2260 encode the SV40 poly(A) sequence; residues 2293-2433 correspond to the 3’ ITR; residues 2508-22963 correspond to FI ori sequences; residues 3350-4210 encode an ampicillin resistance determinant and its signal sequence (residues 3350-3418) expressed by a bla promoter sequence (residues 3245-3349); residues 4381-4969 correspond to the 5’ ITR; residues 20
• a second illustrative rAAV plasmid vector that may be used in accordance with the present invention is pAV-TBG-EGFP (SEQ ID NO:4; Figure 7) which comprises a 5’ ITR, a thyroid hormone-binding globulin (TBG) promoter, a polynucleotide encoding the enhanced green fluorescent protein (EGFP), FLAG-tag and 6xHis-tag sites for facilitating recovery or localization of expressed proteins, an SV40 poly(A) site and a 3’ ITR.
• TAG thyroid hormone-binding globulin
• Plasmid pAV-TBG-EGFP (SEQ ID NO:4):
• residues 1-130 of pAV-TBG-EGFP correspond to the 5’ ITR; residues 150-854 are TBG promoter sequences, with residues 415-824 comprising the TBG promoter; residues 886-1608 encode the EGFP; residues 1630-1653 encode the FLAG-tag; residues 1663-1680 encode the 6xHis-tag; residues 1851-1972 encode the poly(A) sequence; residues 2005-2145 corresponds to the 3’ ITR; residues 2220- 2675 correspond to FI ori sequences; residues 3062-3922 encode an ampicillin resistance determinant and its signal sequence (residues 3062-3130) expressed by a bla promoter sequence (residues 2957-3061); residues 4093-4681 correspond to an ori sequence ( Figure 7).
• the present invention provides a recombinantly-modified adeno- associated virus (rAAV), such as pAV-CMV-EGFP or pAV-TBG-EGFP, that comprises a Cis-Element in one or more of its PI, P2, P3 or P4 Domains, wherein:
• rAAV recombinantly-modified adeno- associated virus
• the PI Domain is 5’ to a 5’ ITR of the rAAV;
• the P4 Domain is 3’ to the 3’ ITR of the rAAV; and wherein the presence of the Cis-Element causes rAAV-producing cells to produce the rAAV at a higher production titer than would be attained with such rAAV if lacking the Cis-Element.
• the Cis-Element will preferably comprise an introduced nucleotide sequence that was not previously present in rAAV vector.
• the introduced nucleotide sequence was previously present in such rAAV plasmid vector, and has been positioned in the recombinantly-produced rAAV plasmid vector, adjacent to, or immediately adjacent to, such previously present nucleotide sequence.
• such introduced nucleotide sequence may be positioned at a site that is not adjacent to such previously present nucleotide sequence.
• an rAAV or an rAAV plasmid vector of the present invention may be envisioned as having, in the 5’ to 3’ direction:
• a“5’ ITR which is an ITR that is located at or near the 5’ end of the polynucleotide chain that comprises the coding strand of the transgene cassette of the rAAV;
• transgene cassette including a preceding (z.e., 5’ -positioned) promoter (“Pro”), the transgene encoding sequence (“Gene”), and following (z.e., 3’ -positioned) poly- A sequence (“Poly A”);
• a“3’ ITR which is an ITR that is located at or near the 3’ end of the polynucleotide chain that comprises the coding strand of the transgene cassette of the rAAV;
• Such PI, P2, P3, and P4 Domains need not all be present in any particular rAAV or rAAV plasmid vector, and an rAAV or rAAV plasmid vector may lack any 1, 2, or 3 of these Domains, or may lack all 4 of such Domains.
• the boundaries of such Domains are defined by the other domains of the rAAV or rAAV plasmid vector.
• the PI Domain extends from the 5’ terminus of the rAAV or rAAV plasmid vector to the 5’ terminus of the 5’ ITR.
• the Pcil site of pAV-CMV-EGFP or pAV-TBG-EGFP is an example of a suitable site within the PI Domain of an rAAV or rAAVplasmid vector for insertion of a Cis-Element.
• the P2 Domain extends from the 3’ terminus of the 5’ ITR to the 5’ terminus of the transgene cassette.
• the P3 Domain extends from the 3’ terminus of the transgene cassette to the 5’ terminus of the 3’ ITR.
• the EcoRl site of pAV-CMV-EGFP or the Spel site of pAV-TBG-EGFP are examples of suitable sites within the P2 Domain of an rAAV or rAAVplasmid vector for insertion of a Cis-Element.
• the P3 Domain extends from the 3’ terminus of the poly(A) sequence to the 3’ ITR of the rAAV or rAAV plasmid vector.
• the Pmll site of pAV- CMV-EGFP or pAV-TBG-EGFP is an example of a suitable site within the P3 Domain of an rAAV or rAAVplasmid vector for insertion of a Cis-Element.
• the P4 Domain extends from the 3’ terminus of the 3’ ITR to the 3’ terminus of the rAAV or rAAV plasmid vector.
• the Kasl site of pAV-CMV-EGFP or pAV-TBG-EGFP is an example of a suitable site within the P4 Domain of an rAAV or rAAVplasmid vector for insertion of a Cis-Element.
• the precise location of a Cis-Element of the present invention within a particular PI, P2, P3, or P4 Domain is not material to the ability of such positioned Cis-Element to mediate an increase in rAAV production titers.
• Insertions of Cis-Element(s) may be made by ligating a Cis-Element into a suitable restriction site or by employing primers to install such Cis-Elements.
• the present invention employs the nomenclature of CisEl, CisE2, etc. to identify particular Cis-Elements. Such designation is followed by“For” or“Rev” in some cases to indicate that the Cis-Element is being (respectively) inserted into the rAAV in its forward orientation or in its reverse orientation. When“For” or“Rev” are not indicated, the Cis-Element is being inserted into the rAAV in its forward orientation. Lastly, the present invention employs the nomenclature PI, P2, P3, or P4 Domain to indicate the domain within which the Cis-Element has been inserted.
• an rAAV or rAAV plasmid vector containing Cis-Element CisEl of the present invention within its PI Domain in its forward orientation is referred to herein by the designation“CisEl-For-Pl” or“CisEl-Pl;” an rAAV or rAAV plasmid vector containing Cis-Element CisE21 of the present invention within its P2 Domain in its forward orientation is referred to herein by the designation“CisE21-For-P2” or “CisEl-P2;” an rAAV or rAAV plasmid vector containing Cis-Element CisE30 of the present invention within its P4 Domain in its reverse orientation is referred to herein by the designation“CisE30-Rev-P4.”
• constructs CisE-Pl, CisE-Rev-Pl, CisE-Pl&P4 CisE- P1-P4 all contain Ci
• such Cis-Elements are actual or potential “G- Quadruplex Sequences” capable of forming a G-Quadruplex Structure.
• the G- Quadruplex Structures of particular relevance to the present invention comprise only a single polynucleotide chain, and have the general formula of four series, each composed of 3 or more deoxyguanosine residues, wherein the first, second and third such series is separated from the fourth such series by from 1 to 7 of any other nucleotide residue.
• a particular polynucleotide will comprise a sequence that is known to comprise a G-Quadruplex Sequence (i.e., an “Actual G-Quadruplex Sequence”).
• a particular sequence will be predicted to comprise a G- Quadruplex Sequence that can form a G-Quadruplex Structure (i.e., a“Potential G- Quadruplex Sequence”).
• Predictive algorithms for determining whether any particular polynucleotide is a potential G-Quadruplex Sequence are well known, and thus the recognition of whether a particular polynucleotide is a potential G-Quadruplex Sequence may be readily accomplished.
• the invention further encompasses compositions such as plasmids that are genetically engineered to replicate high levels of recombinant viral genomes.
• the replication of viral genomes may be regulated through the use of Cis-Elements, including replication origins, promoters and enhancers.
• Cis-Elements can be genetically engineered into recombinant plasmids that are designed to pack AAV vectors.
• the invention encompasses the Cis-Elements can be located before or after ITRs.
• Cis-elements of the present invention that increase AAV production particularly include:
• G-Quadruplex Sequences of wild type AAV genomes e.g, CisEl - CisE16 (SEQ ID NOs:5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35)
• Potential G-Quadruplex Sequences in a reversed orientation e.g, CisEl -Rev - CisE16-Rev (SEQ ID NOs:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36)
• DNA sequences from other viral genomes e.g, CisE27 - CisE30 (SEQ ID NOs: 57, 59, 61 and 63)
• DNA sequences from other viral sources in a reversed orientation e.g, CisE27-Rev - CisE30-Rev (SEQ ID NOs: 58, 60, 62 and 64) and human genomes.
• Table 1 provides the sequences, sequence designations, and origins of such preferred exemplary Cis-Elements of the present invention.
• the inclusion of one or more of the Cis-Elements of the present invention increases rAAV production titers.
• production titer is intended to denote the amount of concentration of infectious rAAV in a preparation. Such amounts or concentrations are preferably determined by titering the AAV or rAAV in such preparation.
• the production titers of the rAAV preparations of the present invention are preferably titered after subjecting producing cells (e.g ., HEK293 transformed with an rAAV plasmid vector, an AAV helper vector providing Rep and Cap proteins, and an Ad helper vector providing required adenovirus transcription and translation factors) to three rounds of freeze / thawing, followed by sonication to release the rAAV particles.
• the preparation is then centrifuged.
• the employed AAV helper vector is localized to the supernatant. An aliquot of the preparation is treated with proteinase K, and the number of AAV genomes is determined.
• an rAAV production titer is said to be“increased” by the methods of the present invention if the production titer obtained from the use of the methods of the present invention is at least 10% greater, more preferably at least 20% greater, still more preferably at least 30% greater, still more preferably at least 40% greater, still more preferably at least 50% greater, still more preferably at least 60% greater, still more preferably at least 70% greater, still more preferably at least 80% greater, still more preferably at least 90% greater, still more preferably at least 2-fold greater, still more preferably at least 110% greater, still more preferably at least 120% greater, still more preferably at least 130% greater, still more preferably at least 140% greater, still more preferably at least 2.5-fold greater, still more preferably at least 160% greater, still more preferably at least 170% greater, still more preferably at least 180% greater, still more preferably at least 190% greater, and still more preferably at least 3- fold greater than the titer obtained from a similarly conducted
• the rAAV whose production titer may be increased using the methods of the present invention may comprise any transgene cassette that permits the rAAV to be packaged into an rAAV plasmid vector that may be encapsidated within an AAV capsid particle.
• transgene cassette(s) may be of human, primate (including chimpanzee, gibbon, gorilla, orangutan, etc), cercopithecine (including baboon, cynomolgus monkey, velvet monkey, etc), canine, glirine (including rat, mouse, hamster, guinea pig, etc) , feline, ovine, caprine, or equine origin.
• such an rAAV or rAAV plasmid vector will encode a protein (e.g. , an enzyme, hormone, antibody, receptor, ligand, etc), or comprise a transcribed nucleic acid, that is relevant to a genetic or heritable disease or condition, such that it may be used in gene therapy to treat such disease or condition.
• a protein e.g. , an enzyme, hormone, antibody, receptor, ligand, etc
• a transcribed nucleic acid that is relevant to a genetic or heritable disease or condition, such that it may be used in gene therapy to treat such disease or condition.
• the methods of the present invention may be used to increase the production titer of rAAV and rAAV plasmid vectors in cells that have been transfected with a desired rAAV or rAAV plasmid vector, and with such one or more viruses and/or helper plasmids that can provide proteins or RNA molecules that are not provided by such rAAV or rAAV plasmid vectors, but are required for their production.
• proteins or RNA molecules include the genes encoding the Rep52 and Rep78 proteins that are required for vector transcription control and replication, and for the packaging of viral genomes into the viral capsule, and, in the case of rAAV, cap genes that encode VP capsid proteins required to form infectious particles.
• Such proteins or RNA molecules also include the viral transcription and translation factors (El a, Elb, E2a, VA and E4) required for AAV proliferation.
• all of these genes and RNA molecules are provided on the same helper virus (or more preferably, helper vector) so as to comprise, in concert with an rAAV, a double plasmid transfection system.
• the required rep and cap genes are provided by one plasmid, and the genes that encode the viral transcription and translation factors are provided on a second plasmid, so that such plasmids, in concert with the rAAV, comprise a triple plasmid transfection system.
• the methods of the present invention may be employed to increase the production titer of rAAV belonging to any serotype, including the AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10 serotypes and the rAAVl, rAAV2, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, and rAAVIO serotypes, and including hybrid serotypes ( e.g ., AAV2/5 and rAAV2/5, which is a hybrid of serotypes 2 and 5 and thus has the trophism of both such serotypes).
• hybrid serotypes e.g ., AAV2/5 and rAAV2/5, which is a hybrid of serotypes 2 and 5 and thus has the trophism of both such serotypes.
• the methods of the present invention may be employed to increase the production titers of rAAV that are to be produced using“helper” RNA or proteins provided by an adenovirus, a herpes simplex virus, a cytomegalovirus, a vaccinia virus or a papillomavirus.
• the methods of the present invention may be employed to increase the production titers of rAAV produced by cells in adherent monolayer culture or in suspension culture, and may be used with any method capable of producing rAAV.
• rAAV is produced by transfecting baby hamster kidney (BHK) cells, or more preferably, human embryonic kidney (HEK) cells grown in tissue culture with the plasmid vectors described above.
• BHK baby hamster kidney
• HEK human embryonic kidney
• the HEK cell line HEK293 (ATCC CRL-1573) and its derivatives, such as HEK293T (ATCC CRL- 3216, which is a highly transfectable derivative of the HEK293 cell line into which the temperature-sensitive gene for SV40 T-antigen was inserted) or HEK293T/17 (ATCC® CRL-11268, which was selected for its ease of transfection) are particularly preferred.
• the HEK293T/17 SF cell line (ATCC ACS-4500) is a derivative of the 293T/17 cell line (ATCC CRL-11268), adapted to serum-free medium and suspension, and may be employed if desired.
• the preferred base medium of the present invention for culturing such cells is Eagle’s Minimum Essential Medium (ATCC Catalog No. 30-2003) or Dulbecco’s Modified Eagle’s Medium (DMEM; Mediatech, Manassas, VA). Fetal bovine serum (e.g ., FBS; HyClone Laboratories, South Logan, UT) is added to a final concentration of 10% in order to make the complete growth medium.
• Eagle’s Minimum Essential Medium and Dulbecco’s Modified Eagle’s Medium are complex media that contain amino acids, vitamins, and optionally glucose, in addition to various inorganic salts.
• the media differ in that Dulbecco's modified Eagle's medium contains approximately four times as much of the vitamins and amino acids present in the original formula of Eagle’s Minimum Essential Medium, and two to four times as much glucose. Additionally, it contains iron in the form of ferric sulfate and phenol red for pH indication (Yao, T et al. (2017)“ Animal-Cell Culture Media: History, Characteristics, And Current Issues,” Reproduc. Med. Biol. 16(2): 99-117).
• Cells to be used for such transfection are preferably passaged twice weekly to maintain them in exponential growth phase.
• an aliquot of, for example, 1 x 10 6 HEK293 or BHK cells per well on a multi-well plate, or 1.5 x 10 7 HEK293 cells per 15-cm dish may be employed.
• HEK293 or BHK cells may be collected from multiple confluent 15-cm plates, and split into two 10-layer cell stacks (Corning, Corning, NY) containing 1 liter of complete culturing medium. In one embodiment, such cells are grown for 4 days in such medium before transfection.
• the two cell stacks may be trypsinized and the cells (e.g., approximately 6 x 10 8 cells) may be resuspended in 200 ml of medium. Preferably, the cells are allowed to attach for 24 hours before transfection. Confluency of the cell stacks may be monitored using a Diaphot inverted microscope (Nikon, Melville, NY) from which the phase-contrast hardware had been removed in order to accommodate the cell stack on the microscope stage.
• a Diaphot inverted microscope Nekon, Melville, NY
• the present invention thus provides a method for increasing the production titer of a recombinantly-modified AAV (rAAV) wherein such method comprises the steps:
• the PI Domain is 5’ to a 5’ ITR of the rAAV;
• the present invention provides a pharmaceutical composition that comprises: (A) a preparation of recombinantly-modified adeno-associated virus (rAAV) that comprise a Cis-Element in one or more of its PI, P2, P3 or P4 Domains, wherein:
• rAAV recombinantly-modified adeno-associated virus
• the PI Domain is 5’ to a 5’ ITR of the rAAV;
• the P2 Domain is 3’ to the 5’ ITR of the rAAV and 5’ to a transgene cassette of the rAAV;
• the P3 Domain is 3’ to the transgene cassette of the rAAV and 5’ to a 3’ ITR of the rAAV;
• the invention additionally includes pharmaceutical compositions that comprise a pharmaceutically acceptable preparation of rAAV produced in accordance with the methods of the present invention, and a pharmaceutically acceptable carrier.
• the rAAV of such pharmaceutical compositions comprises a transgene cassette that encodes a protein, or comprises a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition, and is present in such pharmaceutical composition in an amount effective to (“effective amount”)
• the term“pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
• the term“carrier” refers to a diluent, adjuvant (e.g ., Freund’s adjuvant (complete and incomplete), excipient, or vehicle with which the therapeutic is administered.
• Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
• Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
• suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
• the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
• compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate, or as an aqueous solution in a hermetically sealed container such as a vial, an ampoule or sachette indicating the quantity of active agent.
• a hermetically sealed container such as a vial, an ampoule or sachette indicating the quantity of active agent.
• the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
• an ampoule of sterile water for injection or saline, or other diluent can be provided so that the ingredients may be mixed prior to administration.
• the invention also provides a pharmaceutical pack or kit comprising one or more containers such pharmaceutical composition.
• a pharmaceutical pack or kit comprising one or more containers such pharmaceutical composition.
• Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
• the rAAV of such pharmaceutical compositions is preferably packaged in a hermetically sealed container, such as a vial, an ampoule or sachette indicating the quantity of the molecule, and optionally including instructions for use.
• a hermetically sealed container such as a vial, an ampoule or sachette indicating the quantity of the molecule, and optionally including instructions for use.
• the rAAV of such kit is supplied as a dry sterilized lyophilized powder or water-free concentrate in a hermetically sealed container and can be reconstituted, e.g ., with water, saline, or other diluent to the appropriate concentration for administration to a subject.
• the lyophilized material should be stored at between 2°C and 8°C in their original container and the material should be administered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted.
• the rAAV of such kit is supplied as an aqueous solution in a hermetically sealed container and can be diluted, e.g. , with water, saline, or other diluent, to the appropriate concentration for administration to a subject.
• the kit can further comprise one or more other prophylactic and/or therapeutic agents useful for the treatment of the disease or condition, in one or more containers; and/or the kit can further comprise one or more cytotoxic antibodies that bind one or more cancer antigens associated with cancer.
• the other prophylactic or therapeutic agent is a chemotherapeutic.
• the prophylactic or therapeutic agent is a biological or hormonal therapeutic.
• the methods of the present invention may be used to facilitate the production of rAAV, and may particularly be used to facilitate the production of rAAV that comprise transgene cassettes that encode a protein (e.g ., an enzyme, hormone, antibody, receptor, ligand, etc.), or of rAAV that comprise a transcribed nucleic acid, that is relevant to a genetic or heritable disease or condition, such that it may be used in gene therapy to treat such disease or condition.
• a protein e.g ., an enzyme, hormone, antibody, receptor, ligand, etc.
• rAAV that comprise a transcribed nucleic acid
• achromatopsia ACLM
• alpha-1 antitrypsin AAT
• AADC aromatic L-amino acid decarboxylase
• CHM choroideremia
• BMDSIBM Duchenne muscular dystrophy; dysferlin deficiency; follistatin gene deficiency (BMDSIBM); hemophilia A; hemophilia B; hepatitis A; hepatitis B; hepatitis C; Huntington’s disease; idiopathic Parkinson’s disease; late-infantile neuronal ceroid lipofuscinosis (LINCL, an infantile form of Batten disease); Leber congenital amaurosis (LCA); Leber’s hereditary optic neuropathy (LHON); limb girdle muscular dystrophy IB (LGMD1B); limb girdle muscular dystrophy 1C (LGMD1C); limb girdle muscular dys
• the invention concerns a recombinantly-modified adeno-associated virus (AAV) helper vector that comprises an AAV helper function-providing polynucleotide, and uses and compositions thereof. It is particularly directed to the following embodiments E1-E22:
• rAAV recombinantly-modified adeno-associated virus
• the PI Domain is 5’ to a 5’ ITR of the rAAV;
• the P2 Domain is 3’ to the 5’ ITR of the rAAV and 5’ to a transgene cassette of the rAAV;
• Cis-Element causes rAAV-producing cells to produce the rAAV at a higher production titer than would be attained with such rAAV if lacking the Cis-Element.
• a pharmaceutical composition that comprises:
• rAAV recombinantly-modified adeno-associated virus
• the PI Domain is 5’ to a 5’ ITR of the rAAV;
• the PI Domain is 5’ to a 5’ ITR of the rAAV;
• E5 The recombinantly-modified adeno-associated virus (rAAV) of El, the pharmaceutical composition of E2, or the method of E3, wherein the employed rAAV has been modified to comprise an added Cis-Element in its P2 Domain.
• rAAV adeno-associated virus
• E6 The recombinantly-modified adeno-associated virus (rAAV) of El, the pharmaceutical composition of E2, or the method of E3, wherein the employed rAAV has been modified to comprise an added Cis-Element in its P3 Domain.
• rAAV adeno-associated virus
• E7 The recombinantly-modified adeno-associated virus (rAAV) of El, the pharmaceutical composition of E2, or the method of E3, wherein the employed rAAV has been modified to comprise an added Cis-Element in its P4 Domain.
• rAAV adeno-associated virus
• E8 The recombinantly-modified adeno-associated virus (rAAV) of El, the pharmaceutical composition of E2, or the method of E3, wherein the employed rAAV has been modified to comprise an added Cis-Element in its PI Domain and in one or more of its P2, P3 or P4 Domain.
• rAAV adeno-associated virus
• rAAV adeno-associated virus
• E10 The recombinantly-modified adeno-associated virus (rAAV) of El, the pharmaceutical composition of E2, or the method of E3, wherein the employed rAAV has been modified to comprise an added Cis-Element in its P3 Domain and in its P4 Domain.
• rAAV adeno-associated virus
• rAAV recombinantly-modified adeno-associated virus
• E13 The recombinantly-modified adeno-associated virus (rAAV) of any one of El or E4-E12, the pharmaceutical composition of any one of E2 or E4-E12, or the method of any one of E3-E12, wherein the transgene cassette encodes a protein, or comprises a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition.
• rAAV adeno-associated virus
• rAAV adeno-associated virus
• E15 The recombinantly-modified adeno-associated virus, the pharmaceutical composition, or the method of E14, wherein the employed rAAV belongs to the rAAV2, rAAV5, or rAAV9 serotype, or to a hybrid of the serotypes.
• E16 The recombinantly-modified adeno-associated virus (rAAV) of any one of El or E4-E15, or the method of any one of E3-E15, wherein the cells are human embryonic kidney cells.
• E17 The recombinantly-modified adeno-associated vims (rAAV) of E16, or the method of E16, wherein the cells are human embryonic kidney cells.
• E18 The recombinantly-modified adeno-associated vims (rAAV) of E17, or the method of E17, wherein the are HEK293 cells.
• rAAV adeno-associated vims
• E19 The recombinantly-modified adeno-associated vims (rAAV) of E16, or the method of E16, wherein the cells are baby hamster kidney cells.
• rAAV adeno-associated vims
• E20 The recombinantly-modified adeno-associated vims (rAAV) of E19, or the method of E19, wherein the are BHK21 cells.
• rAAV adeno-associated vims
• E21 The recombinantly-modified adeno-associated vims (rAAV) of E16, or the method of E16, wherein the cells are sf9 insect cells.
• rAAV adeno-associated vims
• E22 The preparation of recombinantly-modified adeno-associated vims (rAAV) of E14, or the pharmaceutical composition of E15, wherein the transgene cassette encodes a protein, or comprises a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition, for use in the treatment of the genetic or heritable disease or condition.
• rAAV adeno-associated vims
• the parent rAAV plasmid vector pAV-TBG-EGFP was modified to contain a cis element within its P2 Domain.
• a series of 27 derivatives of plasmid pAV-TBG-EGFP were constmcted by inserting one of Cis-Element CisEl-CisE27 (Table 1) into the Spel site of the plasmid that is located within the plasmid’s P2 Domain ( Figure 7; Figure 9A).
• the production titers of rAAV obtained using the derivative plasmids in a triple plasmid transfection system ( Figure 2) with the AAV helper plasmid pRC2 providing the AAV rep and cap functions and the Ad helper plasmid pHelper that provided the required adenoviral functions were measured and compared to those obtained with the parental pAV-TBG- EGFP plasmid.
• the rAAV plasmid vector, pAV-TBG- EGFP was modified to contain a Cis-Element within the plasmid’s PI Domain. The effect of that modification on rAAV titer was then assessed as described above.
• a series of 7 derivatives of rAAV plasmid vector pAV- TBG-EGFP were constructed by inserting one of Cis-Element CisEl, CisE20, CisE21, CisE27, CisE28, CisE29, or CisE30 (Table 1) into the Pcil site of the plasmid that is located within the plasmid’s PI Domain ( Figure 7; Figure 10A).
• the production titers of rAAV were obtained essentially as described in Example 1 using a triple plasmid transfection system (Figure 2) with an AAV helper plasmid providing the AAV rep and cap functions (pHelper-Kan) and an Ad helper plasmid that provided the required adenoviral functions (pRC2).
• the production titers were compared with those obtained with the parental pAV-TBG-EGFP plasmid.
• the results of the investigation are shown in Figure 10B, and indicate that all of the Cis-Elements introduced within the PI Domain of pAV-TBG-EGFP resulted in an increase in rAAV production titer.
• the insertion of CisE30 within the PI Domain of the rAAV plasmid vector was found to mediate a 3.0-fold increase in production titer.
• Cis-Element CisE21 Table 1
• Table 1 The effect of the orientation of a Cis-Element in an rAAV plasmid vector on rAAV production titers was investigated by inserting the Cis-Element CisE21 (Table 1) within the PI Domain of the rAAV plasmid vector pAV-TBG-EGFP in either the “forward” orientation (SEQ ID NO:45) or in the“reverse” orientation (SEQ ID NO:46) ( Figure 12A), essentially as described above.
• the production titers of rAAV were obtained essentially as described in Example 1 using a triple plasmid transfection system (Figure 2) with an AAV helper plasmid providing the AAV rep and cap functions and an Ad helper plasmid that provided the required adenoviral functions.
• Figure 13B shows the production titers of rAAV that were obtained using plasmid vector pAV-TBG-EGFP that contained Cis-Element CisE21, CisE22, CisE23, CisE24, CisE25, CisE26, CisE28, CisE29, CisE31, CisE32, CisE33, CisE34, or CisE35 within its PI Domain and Cis-Element CisE30-Rev within its P4 Domain.
• Figure 13C shows the production titers of rAAV that were obtained using plasmid vector pAV-TBG- EGFP that contained Cis-Element CisE21, CisE22, CisE23, CisE24, CisE25, CisE27, CisE28, CisE32, CisE33, or CisE34 within its PI Domain and Cis-Element CisE35- Rev within its P4 Domain.
• the results of the investigation indicate that the presence of two Cis-Elements in the rAAV plasmid vectors synergistically enhanced the increased production titer, relative to that obtained using only a single Cis-Element.

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