Production Of Adeno-associated Virus Vectors Research Articles

Improved Splicing of Adeno-Associated Viral (AAV) Capsid Protein-Supplying Pre-mRNAs Leads to Increased Recombinant AAV Vector Production

Adeno-associated viral (AAV) capsid proteins, thought to be a rate-limiting step in the production of recombinant AAV (rAAV), are translated from spliced mRNAs. Improvement of the native AAV nonconsensus donor sequence increases splicing yet leaves the relative levels of VP1- and VP2/3-encoding mRNAs unchanged, and thus provides a means to increase delivery of correct ratios of AAV capsid proteins. This effect is independent of the AAV serotype used, and occurs whether the rep and cap genes supplied in trans are on the same or separate expression vectors. In the split-vector system, replacement of the more traditionally used cytomegalovirus promoter with that of the AAV5 P41 promoter allowed for even greater levels of splicing, and together with an improved intron donor, led to a 10- to 15-fold increase in the levels of splicing, rAAV production, and transduction compared with levels achieved by traditional cotransfection methods. Thus, the enhancement of splicing presents a useful method to enhance rAAV production via transient transfection.

Intron Splicing–mediated Expression of AAV Rep and Cap Genes and Production of AAV Vectors in Insect Cells

An artificial intron containing the insect cell polyhedrin (polh) promoter was designed, constructed, and inserted into the adeno-associated virus (AAV) Rep and Cap coding sequences to express the Rep and Cap proteins, respectively. The artificial intron was spliced out and full-length Rep78 or VP1 proteins were expressed from the insect promoters located upstream of their respective AUG start codons. The polh promoter located inside the artificial intron was functional, expressed the Rep52 or VP2/VP3 proteins located downstream of the artificial intron, and overlapped with the Rep78 or VP1 proteins. This is the first report that an artificial intron containing an insect cell promoter can be inserted into a coding sequence to express genes with overlapping open-reading frames (ORFs). A method was also established for AAV vector production in insect cells with these intron-containing Rep and Cap coding sequences, and the vectors produced thereby were infectious. These intron-containing AAV Rep and Cap coding sequences were very stable in recombinant baculoviruses and showed no apparent loss of protein expression even after five consecutive amplifications of the plaque-purified recombinant baculoviruses. This newly established AAV production method should prove to be a useful tool for large-scale AAV vector production.

High-titer, serum-free production of adeno-associated virus vectors by polyethyleneimine-mediated plasmid transfection in mammalian suspension cells

Adeno-associated virus (AAV)-based vectors belong to the most promising gene transfer vectors in clinical studies. To provide vector for late-stage clinical trials as well as for a potential commercial phase, a scalable, cGMP-compliant process is required. Nearly all vector production protocols currently approved in Phase I clinical trials rely on AAV production in adherent HEK 293 cells in the presence of serum. In this study, we present a helper- and serum-free production method of AAV vectors in suspension-adapted HEK 293 cells. The method is based on plasmid transfection with 25 kDa linear polyethyleneimine. Compared to existing methods, our system is highly scalable as cells grow in suspension, does not require animal-derived products or the use of an exogenous virus (adenovirus or baculovirus) and yields genomic titers equal to those obtained in adherent HEK 293 cells in the presence of serum. Most importantly, work load and cost could be dramatically reduced in comparison to earlier methods, when comparing the production of equivalent volumes of cell culture media. Thus, our protocol should appeal to both basic research laboratories and cGMP manufacturing units.

84. High Titer Adeno-Associated Viral Vector Production by a Vero-Derived Producer Cell Line

84. High Titer Adeno-Associated Viral Vector Production by a Vero-Derived Producer Cell Line

Production of adeno-associated viral vectors in insect cells using triple infection: Optimization of baculovirus concentration ratios

The production of viral vectors or virus-like particles for gene therapy or vaccinations using the baculovirus expression system is gaining in popularity. Recently, reports of a viral vector based on adeno-associated virus (AAV) produced in insect cells using the baculovirus expression vector system have been published. This system requires the triple infection of cells with baculovirus vectors containing the AAV gene for replication proteins (BacRep), the AAV gene for structural proteins (BacCap), and the AAV vector genome (BacITR). A statistical approach was used to investigate the multiplicities of infection of the three baculoviruses and the results were extended to the production of AAVs containing various transgenes. Highest AAV yields were obtained when BacRep and BacCap, the baculovirus vectors containing genes that code for proteins necessary for the formation of the AAV vector, were added in equal amounts at high multiplicities of infection. These combinations also resulted in the closest ratios of infectious to total AAV particles produced. Overexpression of the AAV structural proteins led to the production of empty AAV capsids, which is believed to overload the cellular machinery, preventing proper encapsidation of the AAV vector transgene, and decreased the viability of the insect cells. Delaying the input of BacCap, to reduce the amount of capsids produced, resulted in lower infectious AAV titers then when all three baculoviruses were put into the system at the same time. The amount of BacITR added to the system can be less than the other two without loss of AAV yield.

Current issues in adeno-associated viral vector production

Adeno-associated virus (AAV) is currently one of the most promising systems for human gene therapy. Numerous preclinical studies have documented the excellent safety profile of these vectors along with their impressive performances in their favored target, consisting of highly differentiated postmitotic tissues such as muscle, central nervous system and liver. Clinical trials have been conducted confirming these data, but also emphasizing the requirement of further high-tech developments of the production and purification procedures that would allow both scaling-up and improvement of vector batch quality, necessary to human application. The scope of this review will be the state of the art in the various production methods of recombinant AAV (rAAV), delimiting their respective perimeter of application and also their main advantages and drawbacks, and thereby shedding light on the main challenges to take in the near future to bring AAV vectors more widely into the clinics.

Adeno-Associated Virus Vectors in Clinical Trials

Adeno-Associated Virus Vectors in Clinical Trials

Adeno-Associated Virus: A Ubiquitous Commensal of Mammals

Adeno-Associated Virus: A Ubiquitous Commensal of Mammals

The adenovirus E1A and E1B19K genes provide a helper function for transfection-based adeno-associated virus vector production.

Although the adenoviral E1, E2A, E4 and VA RNA regions are required for efficient adeno-associated virus (AAV) vector production, the role that the individual E1 genes (E1A, E1B19K, E1B55K and protein IX) play in AAV vector production has not been clearly determined. E1 mutants were analysed for their ability to mediate AAV vector production in HeLa or KB cells, when cotransfected with plasmids encoding all other packaging functions. Disruption of E1A and E1B19K genes resulted in vector yield reduction by up to 10- and 100-fold, respectively, relative to the wild-type E1. Interruption of the E1B55K and protein IX genes had a modest effect on vector production. Interestingly, expression of anti-apoptotic E1B19K cellular homologues such as Bcl-2 or Bcl-x(L) fully complemented E1B19K mutants for AAV vector production. These findings may be valuable for the future development of packaging cell lines for AAV vector production.

Transfection-free and scalable recombinant AAV vector production using HSV/AAV hybrids.

Adeno-associated virus (AAV) vectors are highly efficient tools for use in gene therapy. Current production methods rely on plasmid transfection and are not generally considered amenable to scale-up. To improve recombinant AAV (rAAV) vector production in terms of both final titre and simplicity, we constructed recombinant herpes simplex virus (HSV) vectors, either disabled (ICP27 deleted) or nondisabled, encoding the AAV rep and cap genes. We also integrated an rAAVGFP construct into the nondisabled vector and also into a second pair of HSV vectors (disabled and nondisabled) not expressing rep and cap. Transgene incorporation and expression was confirmed by Southern and Western blot, respectively. Optimal double-infection ratios were established for disabled and nondisabled pairs of rep/cap-expressing and rAAVGFP-containing vectors, resulting in up to 1.55 x 10(12) rAAV capsids and 4 x 10(8) expression units from approximately 1 x 10(7) BHK producer cells. Functionality of the prepared vector was confirmed by the detection of abundant green fluorescent protein (GFP) expression following injections of rAAV preparations into the rat brain. This paper therefore describes a simple, efficient, and transfection-free rAAV production process based on the use of HSV and not relying on a proviral cell line that, with appropriate scale-up, could yield quantities of rAAV sufficient for routine clinical use.

Separate control of Rep and Cap expression using mutant and wild-type loxP sequences and improved packaging system for adeno-associated virus vector production.

Adeno-associated virus (AAV) vectors are a practical choice for gene transfer, and demand for them is increasing. To cope with the necessity in the near future, we have developed a number of approaches to establish packaging cell lines for the production of AAV vectors. In our previous study, a highly regulated expression of large Rep proteins was obtained by using the Cre-loxP switching system. Therefore, in the present study, to regulate Cap expression as well, we developed an inducible expression system for both Rep and Cap proteins by using an additional set of mutant loxP sequences. The mutants possess two base alterations in the spacer region of loxP and recombine specifically with the same counterpart in the presence of Cre. By using two separate plasmids, one with mutant and the other with wild-type loxP sequences, the expression of two different proteins can be induced simultaneously by Cre recombinase. When the LacZ-encoding plasmid vector was used as a packaging model, a significant packaging titer of 2.1 x 1010 genome copies per 10-cm dish was obtained. These results indicate the importance of controlling Cap expression, in addition to Rep, to achieve an optimum production rate for AAV vectors.

From virus evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel vectors for human gene therapy.

Gene transfer vectors based on the human adeno-associated virus serotype 2 (AAV-2) have been developed and tested in pre-clinical studies for almost 20 years, and are currently being evaluated in clinical trials. So far, all these studies have provided evidence that AAV-2 vectors possess many properties making them very attractive for therapeutic gene delivery to humans, such as a lack of pathogenicity or toxicity, and the ability to confer long-term gene expression. However, there is concern that two restrictions of AAV-2 vectors might limit their clinical use in humans. First, these vectors are rather inefficient at transducing some cells of therapeutic interest, such as liver and muscle cells. Second, gene transfer might be hampered by neutralizing anti-AAV-2 antibodies, which are highly prevalent in the human population. In efforts to overcome both limitations, an increasing number of researchers are now focusing on the seven other naturally occurring serotypes of AAV (AAV-1 and AAV-3 to -8), which are structurally and functionally different from AAV-2. To this end, several strategies have been devised to cross-package an AAV-2 vector genome into the capsids of the other AAV serotypes, resulting in a new generation of "pseudotyped" AAV vectors. In vitro and in vivo, these novel vectors were shown to have a host range different from AAV-2, and to escape the anti-AAV-2 immune response, thus underscoring the great potential of this approach. Here the biology of the eight AAV serotypes is summarized, existing technology for pseudotyped AAV vector production is described, initial results from pre-clinical evaluation of the vectors are reviewed, and finally, the prospects of these promising novel tools for human gene therapy are discussed.

Helper virus-free, optically controllable, and two-plasmid-based production of adeno-associated virus vectors of serotypes 1 to 6

We present a simple and safe strategy for producing high-titer adeno-associated virus (AAV) vectors derived from six different AAV serotypes (AAV-1 to AAV-6). The method, referred to as “HOT,” is helper virus free, optically controllable, and based on transfection of only two plasmids, i.e., an AAV vector construct and one of six novel AAV helper plasmids. The latter were engineered to carry AAV serotype rep and cap genes together with adenoviral helper functions, as well as unique fluorescent protein expression cassettes, allowing confirmation of successful transfection and identification of the transfected plasmid. Cross-packaging of vector DNA derived from AAV-2, -3, or -6 was up to 10-fold more efficient using our novel plasmids, compared to a conservative adenovirus-dependent method. We also identified a variety of useful antibodies, allowing detection of Rep or VP proteins, or assembled capsids, of all six AAV serotypes. Finally, we describe unique cell tropisms and kinetics of transgene expression for AAV serotype vectors in primary or transformed cells from four different species. In sum, the HOT strategy and the antibodies presented here, together with the reported findings, should facilitate and support the further development of AAV serotype vectors as powerful new tools for human gene therapy.

Packaging of human chromosome 19-specific adeno-associated virus (AAV) integration sites in AAV virions during AAV wild-type and recombinant AAV vector production.

Adeno-associated virus type 2 (AAV-2) establishes latency by site-specific integration into a unique locus on human chromosome 19, called AAVS1. During the development of a sensitive real-time PCR assay for site-specific integration, AAV-AAVS1 junctions were reproducibly detected in highly purified AAV wild-type and recombinant AAV vector stocks. A series of controls documented that the junctions were packaged in AAV capsids and were newly generated during a single round of AAV production. Cloned junctions displayed variable AAV sequences fused to AAVS1. These data suggest that packaged junctions represent footprints of AAV integration during productive infection. Apparently, AAV latency established by site-specific integration and the helper virus-dependent, productive AAV cycle are more closely related than previously thought.

Rep/Cap gene amplification and high-yield production of AAV in an A549 cell line expressing Rep/Cap.

Cell lines stably expressing rep/cap are important tools for studying adeno-associated virus (AAV) biology and producing AAV vectors. Several rep/cap cell lines have been isolated, each of which is based on HeLa cells. Infection of these cell lines with adenovirus for production of AAV vector is associated with substantial amplification of the rep/cap gene. Concerns over the presence of human papilloma viral (HPV) sequences in HeLa cells may limit use of such lines for production of clinical-grade vectors. Here we describe a non-HeLa-derived rep/cap cell line called K209, generated by stable transfection of A549 cells with a plasmid construct containing the P5 rep/cap cassette from AAV2. Infection of K209 cells with adenovirus leads to a 1000-fold amplification of the rep/cap gene with high-yield production of AAV vectors. The multiplicity of infection (MOI) of adenovirus that led to maximum amplification of the rep/cap gene and high-level production of AAV is 10 times higher in the HeLa-based cell line than that required in K209 cells. Our data suggest that papilloma-derived gene products present in HeLa cells are not required for high-yield production of AAV vectors.

Recent advances in recombinant adeno-associated virus vector production

Adeno-associated virus (AAV) is a replication-defective parvovirus that is being developed as a vector for human gene transfer. Recombinant AAV (rAAV) vectors are being proposed as a gene transfer vehicle for an array of human diseases. The recent interest in rAAV has been driven by the unexpected finding that these simple vectors can efficiently transduce a variety of postmitotic cells, resulting in long-lived, robust gene expression. However, a major obstacle to commonplace usage of rAAV vectors was the production in sufficient quantities for preclinical and human trials. Fortunately, several recent technological advances in vector production, purification, and titration have resulted in significant increases (>10-fold) in production capacity. Thus, there are several methods for the production of rAAV in excess of 10(4) particles/cell, levels that should permit widespread use of this technology for clinical applications.

23] Recombinant adeno-associated viral vector production using stable packaging and producer cell lines

23] Recombinant adeno-associated viral vector production using stable packaging and producer 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.

Adeno-associated virus-based vectors in gene therapy.

Adeno-associated virus (AAV) vectors were shown capable of high efficiency transduction of both dividing and nondividing cells and tissues. AAV-mediated transduction leads to stable, long-term transgene expression in the absence of apparent immune response. These properties and the broad host range of AAV vectors indicate that they constitute a powerful tool for gene therapy purposes. An additional potential benefit of AAV vectors is their ability to integrate site-specifically in the presence of Rep proteins which can be expressed transiently, thus limiting their suspected adverse effects. The major restrictions of AAV as vectors are their limited genetic capacity and strict packaging size constraint of less than 5 kb. Another difficulty is the labor-intensive and expensive procedure for the production and packaging of recombinant AAV vectors. The major benefits and drawbacks of AAV vectors and advances made in the past 3 years are discussed.

Packaging cells based on inducible gene amplification for the production of adeno-associated virus vectors.

Although vectors based on adeno-associated virus (AAV) offer several unique advantages, their usage has been hampered by the difficulties encountered in vector production. In this report, we describe a new AAV packaging system based on inducible amplification of integrated helper and vector constructs containing the simian virus 40 (SV40) replication origin. The packaging and producer cell lines developed express SV40 T antigen under the control of the reverse tetracycline transactivator system, which allows inducible amplification of chromosomal loci linked to the SV40 origin. Culturing these cells in the presence of doxycycline followed by adenovirus infection resulted in helper and vector gene amplification as well as higher vector titers. Clonal producer cell lines generated vector titers that were 10 times higher than those obtained by standard methods, with approximately 10(4) vector particles produced per cell. These stocks were free of detectable replication-competent virus. The lack of a transfection step combined with the reproducibility of stable producer lines makes this packaging method ideally suited for the large-scale production of vector stocks for human gene therapy.