Production Of rAAV Vectors Research Articles

OneBac 2.0: Sf9 Cell Lines for Production of AAV5 Vectors with Enhanced Infectivity and Minimal Encapsidation of Foreign DNA.

Scalable production of recombinant adeno-associated virus vectors (rAAV) in baculovirus-infected Sf9 cells yields high burst sizes but variable infectivity rates per packaged AAV vector genome depending on the chosen serotype. Infectivity rates are particularly low for rAAV5 vectors, based on the genetically most divergent AAV serotype. In this study we describe key improvements of the OneBac system for the generation of rAAV5 vectors, whose manufacturing has been unsatisfactory in all current insect cell-based production systems. The Sf9 cell-based expression strategy for AAV5 capsid proteins was modified to enhance relative AAV5 VP1 levels. This resulted in a 100-fold boost of infectivity per genomic AAV5 particle with undiminished burst sizes per producer cell. Furthermore, the issue of collateral packaging of helper DNA into AAV capsids was approached. By modifications of the AAV rep and cap expression constructs used for the generation of stable Sf9 cell lines, collateral packaging of helper DNA sequences during rAAV vector production was dramatically reduced down to 0.001% of packaged rAAV genomes, while AAV5 burst sizes and infectivity rates were maintained. OneBac 2.0 represents the first insect cell-based scalable production system for high per-particle AAV5 infectivity rates combined with minimal collateral packaging of helper DNA, allowing the manufacturing of safe AAV5-based gene therapies for clinical application.

557. Impact of the Integrity of the ITRs on the Production of rAAV Vectors

The Adeno-Associated Virus's (AAV) Inverted Terminal Repeats (ITRs) are key features of the AAV. ITRs are implied in the replication, encapsidation of the AAV genome, along with its integration in the host genome and its excision. These sequences are the only AAV DNA sequences conserved in the recombinant AAV as they allow its replication, encapsidation and long-term maintenance in the host non-dividing target cells. The ITRs are highly GC rich and structured due to the presence of repetitive palindromic sequences.During its replication, the AAV is able to reconstitute incomplete or imperfect ITR sequences. Thus it is often found that rAAV genomes cloned into the backbones of the DNA plasmids used for production, have imperfect ITRs sequences which are shortened or contain mutations.In this work, we study the impact of those imperfect ITRs and their impact on the levels of encapsidated rAAV genomes and cellular/baculovirus contaminating DNA sequences, in the baculovirus/Sf9 cells production system.

Herpes simplex virus clearance during purification of a recombinant adeno-associated virus serotype 1 vector.

Gene delivery vectors based on adeno-associated virus (AAV) have potential utility for treatment of many genetic disorders. Current AAV vector manufacturing methods employ helper viruses to deliver functions needed to produce replication-defective recombinant AAV (rAAV) vectors, and clearance of infectious helper virus from the drug substance is essential for ensuring the safety of rAAV-based therapies. We have developed a manufacturing method for the production of rAAV vectors using a recombinant herpes simplex virus type 1 (rHSV) complementation system in suspension baby hamster kidney cells. The manufacturing process includes three primary unit operations, detergent lysis of the cell harvest and two downstream column chromatography steps, which achieve viral clearance. These unit operations inactivate and remove HSV, including replication-competent HSV present at low levels in rHSV helper stocks. Here we report results quantifying the reduction in HSV achieved during rAAV vector purification. Clearance of HSV was at least 6.84 log10 with 1% Triton X-100, 4.34 log10 with CIM Q column chromatography, and 2.86 log10 with AVB affinity chromatography. Combined, these three orthogonal methods achieved clearance of at least 14.04 log10 of HSV. The total input quantity of rHSV in a 100-liter production batch is approximately 1.2×10(12) plaque-forming units (pfu), and after purification, the concentration of residual rHSV in the resulting drug substance of approximately 450 ml would be less than 2.42×10(-5) pfu/ml. A rAAV vector produced using this method was used in a clinical trial in which subjects receive up to 100 intramuscular injections of 1.35 ml each, which would contain a maximum of 3.27×10(-3) pfu of HSV. These results support the safety of rAAV vectors produced using our rHSV complementation method.

A Simplified Baculovirus-AAV Expression Vector System Coupled With One-step Affinity Purification Yields High-titer rAAV Stocks From Insect Cells

Scalable methods of recombinant adeno-associated virus (rAAV) production have gained much recent interest as the field of rAAV-mediated gene therapy approaches the clinic. In particular, the production of rAAV vectors in insect cells via the use of recombinant baculovirus technology has proven to be an efficient and scalable means of rAAV production. Here, we describe a method for the production of rAAV serotypes 1 and 2 in insect cells using a simplified baculovirus-AAV expression vector system coupled with particle purification via affinity chromatography. The number of separate baculovirus constructs required for rAAV production was reduced by genetically modifying the AAV rep gene to allow expression of the AAV-encoded replication enzymes, Rep78 and Rep52, from a single mRNA species and combining the modified rep gene with an AAV cap gene expression cassette in a single baculovirus construct. Additionally, we describe lysis, binding, and elution conditions compatible with a commercially available affinity medium (AVB Sepharose High Performance) used to purify rAAV particles to near homogeneity in a single chromatography step. Using the described method, we obtained an average yield of 7 x 10(4) purified rAAV particles per cell (range: 3.7 x 10(4) to 9.6 x 10(4)) from suspension cultures of recombinant baculovirus-infected insect cells.

Improved splicing of the AAV capsid protein-supplying pre-mRNAs leads to increased rAAV vector production

Improved splicing of the AAV capsid protein-supplying pre-mRNAs leads to increased rAAV vector production

503. A Novel Herpes Simplex Virus Helper Based Production of Adeno- Associated Virus Vectors for Treatment of Retinal Angiogenesis

AGTC's proprietary rAAV vector production method uses recombinant herpes simplex virus (HSV) as the helper agent for rAAV production. This production method has been demonstrated to be both highly scalable and productive compared to other rAAV production methods such as the transient co-transfection or adenovirus co-infection process. rAAV production by rHSV-1 methodology requires two recombinant HSV-1 vectors, rHSV-1- rep2cap2 which expresses the AAV 2 rep and cap genes, and a second rHSV-1 that contains the therapeutic gene of interest flanked by the AAV-2 inverted terminal repeats.Both rHSV vectors are deleted in an essential gene for HSV replication and hence they are propagated only in a complementing cell line. Here we report the construction and characterization of rHSV-TR-shFlt-1 that contains a novel soluble hybrid version of Flt-1, the receptor for vascular endothelial growth factor (VEGF). This novel shFlt-1 is a high-affinity binder of VEGF, a central player in pathologic neovascularization in both wet age-related macular degeneration (AMD) and proliferative diabetic retinopathy (PDR). To construct rHSV-TR-shFlt-1, a shuttle plasmid containing the expression cassette of sFlt-1 flanked with homologous HSV-1 sequences was first constructed and co-transfected with rHSVd27.1- TR-GFP viral DNA into complementing V27 cells. rHSV-TR-shFlt-1 was screened against the parental rHSV-1-GFP viruses that express fluorescent GFP, and the homologous recombination was further confirmed by Southern blot analysis. Selected rHSV-TR-shFlt-1 clones were propagated through 10 passages and their ability to produce rAAV2-TR-shFlt-1 vector was examined by real-time PCR. The expression level, size, and in-vitro biological activity of sFlt-1 expressed in rAAV2-TR-shFlt-1 transduced cells were determined by ELISA, immunoblot, and the HUVECs proliferation assay respectively. Stability studies indicated that the rHSV-TR-shFlt-1 helpers maintained their ability to produce rAAV comparable to that of the positive control rHSV-1-GFP through 10 passages. Preliminary data from the oxygen-induced retinopathy (OIR) mouse model suggests that rAAV2-shFlt-1 derived from rHSV1- TR-shFlt-1 is able to inhibit retinal neovascularization, and there is no significant difference compared to rAAV2-shFlt-1 vector made by the transfection method. To our knowledge this is the first report of the construction and characterization of a recombinant HSV helper virus to produce a rAAV vector encoding a therapeutic gene for inhibition of ocular angiogenesis. Further preclinical evaluation of the AAV2-shFlt-1 is in the process.

Successful Production of Pseudotyped rAAV Vectors Using a Modified Baculovirus Expression System

Scalable production of rAAV vectors remains a major obstacle to the clinical application of this prototypical gene therapy vector. A recently developed baculovirus-based production protocol (M. Urabe et al., 2002, Hum. Gene Ther. 13, 1935–1943) found limited applications due to the system's design. Here we report a detailed analysis of the stability of the original baculovirus system components BacRep, BacVP, and transgene cassette-containing BacGFP. All of the baculovirus helpers analyzed were prone to passage-dependent loss-of-function deletions resulting in considerable decreases in rAAV titers. To alleviate the instability and to extend the baculovirus platform to other rAAV serotypes, we have modified both Rep- and Cap-encoding components of the original system. The modifications include a parvoviral phospholipase A2 domain swap allowing production of infectious rAAV8 vectors in vivo. Alternatively, an infectious rAAV8 (or rAAV5) vector incorporating the AAV2 VP1 capsid protein in a mosaic vector particle with AAV8 capsid proteins was produced using a novel baculovirus vector. In this vector, the level of AAV2 VP1 expression is controlled with a “riboswitch,” a self-cleaving ribozyme controlled by toyocamycin in the “ON” mode. The redesigned baculovirus system improves our capacity for rAAV manufacturing by making this production platform more applicable to other existing serotypes.

400. Successful Production of Pseudotyped Mosaic rAAV Vectors Using a Modified Baculovirus Expression System

Top of pageAbstract Scalable production of rAAV vectors remains a major obstacle to the clinical application of this protypical gene therapy vector. A recently developed baculovirus-based production protocol (Urabe et al) found limited applications due to the system's design. Here we report a detailed analysis of the stability of the original baculovirus system components BacRep, BacVP, and transgene cassette-containing BacGFP. All of the baculovirus helpers analyzed were prone to passaging-dependent, loss-of-function deletions resulting in considerable decreases in rAAV titers. To alleviate the instability problem, we have modified the Rep-encoding component by splitting it into two separate vectors. Furthermore, we have examined the expression limits of the remaining components of this system in order to optimize its application to AAV vector production. In addition, to successfully employ this system to pseudotyped AAV vectors, we have introduced a novel modular approach of parvoviral phospholipase A2 (PLA2) domain swapping that allows for baculovirus production of infectious AAV8 based vectors. The novel chimeric rAAV2/8 vector, produced in Sf9 cells, incorporated AAV2 PLA2 into AAV8 capsid structure and was characterized by robust transduction in vivo. Finally, using the novel riboswitch-controlled gene expression system, we have constructed a universal Bac helper vector for producing infectious mosaic rAAV vector of any given serotype, as demonstrated for rAAV5 and rAAV8. The re-designed baculovirus system improves our capacity for rAAV production by making this AAV platform more applicable to other existing serotypes.

420. Adaptation of Baculovirus System for Large-Scale Production of rAAV Vector Serotypes

Scalable production of rAAV vectors is a technically challenging practice. A recently developed baculovirus-based production protocol (Urabe et al), although potentially promising and elegant, proved to be inconsistent and unreliable. Here we report the modification of the original baculovirus system that is amenable to scale-up and suitable for the production of other AAV serotypes similar to conventionally produced vectors. Both Rep and Cap components of the original system have been modified. The Rep52 and Rep78 transcription units of the original BacRep vector were placed onto two separate vectors thereby greatly enhancing the stability of the rep genes for at least 5 continuous passages. In addition, the capsid genes of the BacVP helper were modified by substituting AAV2 VP1 phospholipase A2 (PLA2) domain for the homologous region in the other serotype baculovirus helpers (for example the AAV8 serotype). The resulting chimeric AAV2/8 VP1 reconstituted the wt AAV2 enzymatic PLA2 activity and, consequently, wt AAV8 infectivity. Examples of scale-up production and experiments in vivo will be discussed in detail.

126. Production of rAAV Vectors with Increased Potency from Stable Cell Lines

126. Production of rAAV Vectors with Increased Potency from Stable Cell Lines

Development and Characterization of an Antisense-Mediated Prepackaging Cell Line for Adeno-Associated Virus Vector Production

One of the limitations of recombinant adeno-associated virus (rAAV) vector systems for gene therapy applications has been the difficulty in producing the vector in sufficient quantity for adequate evaluation. Since the AAV Rep proteins are cytotoxic, it is not easy to establish stable cell lines that express them constitutively. We describe a novel 293-derived prepackaging cell line which constitutively expresses the antisense rep/cap driven by a loxP-flanked CMV promoter. This cell line was converted into a packaging cell line expressing Rep/Cap for rAAV vector production through adenovirus-mediated introduction of a Cre recombinase gene. Without the introduction of the Cre recombinase gene, the cell line was shown to produce neither Rep nor Cap. rAAV vector was produced (1 × 109 genome copies/3.5-cm dish) 4 days after the transduction with Cre-expression adenovirus vector together with transfection of AAV vector plasmid. We further showed that the addition of Cap-expression adenovirus vector caused a 10-fold increase in the yield of rAAV vector. This system is also capable of producing rAAV as a transfection-free system by using a small amount of rAAV instead of vector plasmid.

Generation of recombinant adeno-associated virus vectors by a complete adenovirus-mediated approach.

An efficient approach to the large-scale production of rAAV will significantly facilitate the application of this promising gene delivery vector in human gene therapy applications. In this study, we report a novel approach to rAAV production that is based exclusively on the adenovirus vector, without the need for plasmid transfections and special packaging cell lines. All components required for rAAV production, including the rep and cap genes, and the therapeutic gene(s) are delivered to the widely used 293 packaging cell line by adenovirus vectors. High-titer rAAV vectors (200-600 infectious units/producer cell) were obtained by use of this approach. As adenovirus vectors can be produced to high titers and they can infect cells in suspension efficiently, this approach may be amenable to scaled-up production of rAAV vectors.

Production of Recombinant Adeno‐Associated Viral Vectors

AbstractRecombinant adeno‐associated viral (rAAV) vectors are capable of delivering genes to both dividing and nondividing cells. This property, along with the physical stability of the virions, permits transduction of cells both in tissue culture and in the whole animal. In this unit, protocols for the adenovirus‐free production of rAAV vectors, their purification, and their quantitation are provided. The production and purification of the rAAV vectors are described, using two different methods. Two assays for determining rAAV titers are also presented: dot blot and in vitro infection with a transgene expression assay.

High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap.

Recombinant adeno-associated virus type 2 (rAAV) vectors have recently been used to achieve long-term, high level transduction in vivo. Further development of rAAV vectors for clinical use requires significant technological improvements in large-scale vector production. In order to facilitate the production of rAAV vectors, a recombinant herpes simplex virus type I vector (rHSV-1) which does not produce ICP27, has been engineered to express the AAV-2 rep and cap genes. The optimal dose of this vector, d27.1-rc, for AAV production has been determined and results in a yield of 380 expression units (EU) of AAV-GFP produced from 293 cells following transfection with AAV-GFP plasmid DNA. In addition, d27.1-rc was also efficient at producing rAAV from cell lines that have an integrated AAV-GFP provirus. Up to 480 EU/cell of AAV-GFP could be produced from the cell line GFP-92, a proviral, 293 derived cell line. Effective amplification of rAAV vectors introduced into 293 cells by infection was also demonstrated. Passage of rAAV with d27. 1-rc results in up to 200-fold amplification of AAV-GFP with each passage after coinfection of the vectors. Efficient, large-scale production (>109 cells) of AAV-GFP from a proviral cell line was also achieved and these stocks were free of replication-competent AAV. The described rHSV-1 vector provides a novel, simple and flexible way to introduce the AAV-2 rep and cap genes and helper virus functions required to produce high-titer rAAV preparations from any rAAV proviral construct. The efficiency and potential for scalable delivery of d27.1-rc to producer cell cultures should facilitate the production of sufficient quantities of rAAV vectors for clinical application.

Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses.

Gene transfer vectors based on the replication-defective human parvovirus, adeno-associated virus type 2 (AAV-2), are viable candidates for in vivo and ex vivo human use. However, widespread testing of AAV vectors has been limited by difficulties in generating pure, high-titer vector stocks that are fully characterized. To address these issues, we have developed a single-step purification scheme using heparin affinity chromatography. Recovery from the crude lysate starting material exceeds 70%, and the end product rAAV vector is highly purified and appears to be free of adenovirus and cellular contaminates. Importantly, purified vectors retain predicted in vivo biologic activity. Concurrently, we have developed simple and rapid approaches for vector quantification using real-time PCR. These new methods, combined with the use of stable producer cell lines for rAAV production, make the commercial production of rAAV vectors for human use truly viable and pragmatic.

Production of recombinant adeno-associated virus vectors using a packaging cell line and a hybrid recombinant adenovirus.

Recombinant adeno-associated virus (rAAV) vectors are under consideration for a wide variety of gene therapy applications. One of the limitations of the rAAV vector system has been the difficulty in producing the vector in sufficient quantity for adequate preclinical and clinical evaluation. A common method for vector production involves large-scale transient transfection of multiple plasmids into cultured cells. Because this approach might not be feasible for clinical scale manufacturing, we have sought approaches for rAAV vector production that avoid transient transfection procedures. In previously reported work, we generated an AAV packaging cell line that produces infectious rAAV when the vector genome is transfected into the cell line as plasmid DNA. We have now extended this approach by constructing a hybrid recombinant adenovirus (rAd) that contains a complete rAAV vector genome in the E1 region. This hybrid virus is used to deliver the rAAV genome to the packaging cell line (in the place of plasmid transfection). rAAV is produced when the packaging cell line is infected with the hybrid adenovirus and wild-type adenovirus. This method avoids the need for plasmid transfection and is adaptable to large-scale manufacturing processes.

A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle.

Adeno-associated virus (AAV) replication is dependent on two copies of a 145-bp inverted terminal repeat (ITR) that flank the AAV genome. This is the primary cis-acting element required for productive infection and the generation of recombinant AAV (rAAV) vectors. We have engineered a plasmid (pDD-2) containing only 165 bp of AAV sequence: two copies of the D element, a unique sequence adjacent to the AAV nicking site, flanking a single ITR. When assayed in vivo, this modified hairpin was sufficient for the replication of the plasmid vector when Rep and adenovirus (Ad) helper functions were supplied in trans. pDD-2 replication intermediates were characteristic of the AAV replication scheme in which linear monomer, dimer, and other higher-molecular-weight replicative intermediates are generated. Compared to infectious AAV clones for replication, the modified hairpin vector replicated more efficiently independent of size. Further analysis demonstrated conversion of the input circular plasmid to a linear substrate with AAV terminal repeat elements at either end as an initial step for replication. This conversion was independent of both Rep and Ad helper genes, suggesting the role of host factors in the production of these molecules. The generation of these substrates suggested resolution of the modified terminal repeat through a Holliday-like structure rather than replication as a mechanism for rescue. Production of replicative intermediates via this plasmid substrate were competent not only for AAV DNA replication but also for encapsidation, infection, integration, and subsequent rescue from the chromosome when superinfected with Ad and wild-type AAV. These studies demonstrate that this novel 165-bp ITR substrate is sufficient in cis for the AAV life cycle and should provide a valuable reagent for further dissecting the cis sequences involved in AAV replication, packaging, and integration. In addition, this novel plasmid vector can be used as a substrate for both rAAV vector production and synthetic plasmid vector delivery.