Ruisai small classroom technology articles 0702-1313
Summary:
Adeno-associated virus (AAV) is a human parvovirus. Â Because it can be widely used as a gene therapy vector. Most current protocols for generating rAAV require co-transfection of a vector plasmid and a packaging plasmid expressing viral replication and structural genes into adenovirus (Ad) infected cultured cells. However , the need for Ad co-transfection can also be ruled out by the new helper plasmids (pH 3 and pH 5). The helper plasmid expresses the rep and cap genes of AAV and the Ad E2A, VAI and E4 genes. When the helper plasmid was co-transfected into human 293 cells without Ad infection, the rAAV vector yield was 80 times greater than the pAAV/Ad packaging plasmid. In addition, replication-capable AAV was less than 0.00125% during rAAV preparation. The dual plasmid transfection system enables the AAV vector system to be more widely used due to its simplicity and high yield.
1. Introduction
Adeno-associated virus (AAV) is a common human parvovirus that is naturally deficient, uncoated and pathogenic. The AAV replication cycle consists of two distinct phases: the incubation period and the proliferative phase. In the absence of a helper virus such as adenovirus (Berns, 1990; Muzyczka, 1992; Berns and Giraud, 1996), herpes virus, vaccinia virus or under genotoxic conditions (Yakobson et al., 1989), AAV is capable of replicating to produce progeny virus particles. In the absence of a helper virus, the adeno-associated virus integrates its genome into a specific site on chromosome 19 and remains integrated until the subsequent helper virus rescues it from the latent state (Kotin et al., 1990). The site-specific integration ability of AAV, its natural defects, and its lack of pathogenicity make it possible to become a gene therapy vector. The AAV genome is a linear, single-stranded (ssDNA) molecule of 4680 nucleotides with a 145 base end repeat (TR) at each end (Srivastava et al., 1983). The TR sequence is folded into a hairpin structure as the only known cis-acting element required for DNA replication initiation and packaging of the recombinant AAV genome for infectious viral particles.
Construction of recombinant adenoviral vectors (rAAV) typically involves the cleavage of the rep and cap genes and the insertion of the transgene of interest between the TR elements. Key Ad-assisted genes include: E1a transcriptionally activates Ad and AAV genes, E1b and E4 encode proteins that promote mRNA shipment to the cytoplasm, E2a expresses a ssDNA-binding protein to promote AAV DNA replication, and VAI RNA generates a small RNA transcript to enhance AAV capsid Translation of mRNA.
The limitations of this cumbersome process prevented the widespread development of rAAV as a vector for gene therapy. The formation of the support as described above always results in significant Ad contamination of the support, thus requiring heat treatment and stringent purification methods to inactivate and remove Ad virus particle contamination. Although there are usually no homologous sequences between the vector and the packaging plasmid, wild-type replication competent AAV (rcAAV) is often produced (Allen et al, 1997; Wang et al, 1998). Many attempts have been made to improve the packaging efficiency of rAAV vectors. These include: development of cell lines that express some or all of the AAV genes required for packaging (Yang et al, 1994; Clark et al, 1995; Tamayose et al, 1996; Inoue and Russell, 198), construction of Ad helper plasmids can be used to replace Ad infections (Matsu***a et al, 1998; Xiao et al, 1998b) and development of recombinant Ad carrying the rAAV vector genome (Gao et al, 1998).
We have constructed a new helper plasmid that does not require infection with Ad during rAAV packaging. These plasmids contain the VA, E2a and E4 genes of the Ad genome and the rep and cap genes of AAV. When transfected into human 293 cells containing the Ad5E1a and E1b genes, the helper plasmid provides an efficient, Ad-free contamination packaging system. The rAAV titers generated by this new system were 80-fold higher than most of the pAAV/Ad packaging plasmids (Samulski et al., 1989). In addition, Ad can no longer be produced, and AAV with replication ability has not been found in any vector preparation process.
2. Materials and methods
2.1. Plasmids, cells and viruses
An rAAV vector plasmid, pTR-UF5 expressing human green fluorescent protein (Peel et al., 1997) was used in all packaging experiments to optimize the system. This pAVbgal vector plasmid contains AAV TR, which is located on both sides of the cytomegalovirus (CMV) early transcription promoter and the E. coli b galactosidase gene. The PAAV/Ad plasmid was used as a packaging plasmid for comparison with a helper plasmid (Samulski et al., 1989). The pCDMrep plasmid contains the AAVrep gene and is regulated by the CMV early promoter (Yang and Trempe, 1993).
The 19193 bp helper plasmid, pSH3 and pSH5, can be constructed in several steps. A 1.5 kb fragment of HindIII to SalI containing the VAI and VAII genes (nt9831-11555 from Ad5) was inserted into the same site of pGEM3Z to generate pVA3. A 5.8 kb fragment of BamHI and EcoRI containing the E2a gene (nt21563-27331 from Ad) was inserted into the same site of pVA3 to generate pVA3E2a. The 4.3 kb XbaI fragment from pSub201 (nt177-4471 in AAV2), containing the AAV rep and cap genes, was subsequently inserted into the two XbaI sites of pVAE2aE4. One of the XbaI sites on pVAE2aE4 was located in the E2a and VAI and II gene segments, and the other site (nt10580 in the Ad2 genome) was found upstream of the VAI gene. The two versions of the helper plasmids (pSH3 and pSH5) differ in the orientation of the 4.3 kb XbaI insert and are shown in Fig. 1B. The pSH3 and pSH5 helper plasmids were stored in the HB101 strain of E. coli. The plasmid was purified from 500 ml of bacterial culture and divided into 4 Ì 125 m, each fraction was treated with the Qiagen Plasmid Maxi kit. Human 293 (Ad-5 transformed fetal kidney epithelial) cells and HeLa cells were cultured in EMEM supplemented with 10% fetal bovine serum and antibodies. The cells were cultured in a monolayer medium at 37 ° C, 5% CO 2 . Control packaging experiment Ad5 type with a fold of infection (moi) of 15 was cultured in serum-free medium for 1 h, followed by transfection. Ad titration by plaque formation on HeLa cells. Fig. 1. Plasmid used for rAAV packaging (A) pTR-UF5 contains the human gfp gene driven by the CMV promoter (arrow) and the neoR gene driven by the HSV-tk promoter (arrow). There are AAV TR elements (filled squares) on either side of the entire build. pAVbgal contains the E. coli b-galactosidase gene and is under the control of the CMV promoter (arrow). On both sides of the transcription cassette are AAV TR elements (filled squares). pAAV/Ad contains the AAV rep and cap genes, flanked by Ad-terminal repeat elements (empty squares) (B) pSH3 and pSH5 contain Ad E4, E2a and VA RNA genes. Both plasmids also contain the AAV rep and cap genes. The numbers under the figure refer to the number of nucleotides in the Ad and AAV genomes. The difference between pSH3 and pSH5 is the orientation in the AAV DNA sequence. For the sake of clarity, the rest of the plasmid map is not listed.
2.2. rAAV vector production and titration For small-scale packaging experiments, 8 Ì105 293 cells were grown on 35 mm 6 well plates. After 24 hours, the cultures were transfected in triplicate with lipofectamin (Life Technologies) according to the manufacturer's protocol. For the initial optimization experiments, the ratio of vector to helper plasmid used is listed in Table 1. Depending on the size of the vector and the helper plasmid, the 1 mg:3 mg ratio corresponds to a molar ratio of pTR-UF5 to pSH3/5 of 1:1. The pAAV/Ad plasmid was co-infected with Ad, and 3 or 5 mg of pAAV/Ad was co-infected with 1 mg of pTR-UF5. The pGEM3Z plasmid (Promega) was co-transfected to maintain the same total DNA concentration for different transfections. For transfection with Ad infection, the virus was adsorbed to the cell monolayer in serum-free medium for 1 h before DNA was added to the cells. 72 hours after transfection, the cells were scraped into 2 ml of medium and harvested. The cells were then washed with 1 ml sterile PBS. The PBS washing solution was added to the cell suspension, and the cells were sonicated (5, 36 mm, 45 s) after freezing and thawing for 5 times. The extract was centrifuged at 4000 Ìg for 10 minutes to remove cell debris. The lysate supernatant contained the recombinant vector vAVgfp, which was used in subsequent transduction. These transfected Ads were heat treated at 56 ° C for 30 minutes prior to transduction. For large-scale vector packaging, 20 Ì1 125px plates, each containing 5 Ì106 293 cells, and transfected with 10 mg pAVgal and 50 mg pSH3 by calcium phosphate precipitation method (Wigler et al., 1979). After 72 hours, harvest The culture was pelleted by low-speed centrifugation, frozen and thawed 4 times, and disrupted by sonication as described above. CsCl2 was added to the lysate at a final concentration of 1.41 g/ml, determined by refractive index. The suspension was centrifuged at 40 000 rpm for 48 hours on a SW41 rotor. The strip containing the visible carrier is removed from the gradient and its density can be determined by measuring the index of refraction. The CsCl2 centrifugation step was repeated, and the obtained vector was prepared for dialysis, and the PBS was replaced 4 times and stored at -80 °C. The resulting vector vAVgal was titrated as follows. To determine the titer of rAAV, 4 Ì104 HeLa cells were plated into 24-well plates and infected with Ad with a moi of 10, transduced with serially diluted transfected cell lysates. After 2 days, the cytopathic effect was found to be observed at 20 times using a DIAPHOT-*** Ricoh inverted microscope with an EPI-fluorescence device. Counting produces a dilution factor of a well-isolated fluorescent cell vector. Thus, each positive cell represents a vector transduction unit (TU). To determine the titer of the rAAVgal vector, a dilution of the purified vector was used to infect the cells. The cells were fixed with 95% methanol dissolved in PBS and developed with X-gal. Each blue-stained cell represents a vector transduction unit (TU). Vector genome copy number was determined by dot blot hybridization analysis and A260 was determined as previously described (Fisher et al., 1996). For small scale packaging experiments on 6-well plates, the prepared original vector was treated with DNase I for 30 minutes at 37 °C. DNase was inactivated at 75 ° C and proteins were digested with 50 mM Tris 1 mM EDTA 0.1% SDS 0.5 mg/ml proteinase K for 12 hours at 37 °C. For the CsCl2 purified vector, the same procedure was used, but without the DNase step. The vector DNA was boiled for 5 minutes and applied to a nitrocellulose membrane (Schleicher and Schuell) using a Bio-Dot apparatus (Bio-Rad). Each well was washed with 300 M of 0.5 M ammonium acetate (pH 5.2). Wash blots were processed according to the 2.4 part Southern blot method, vector specific probe labels, and exposed to Kodak X-omat X-ray film (Eastman Kodak). The vector hybridization signal was compared by comparing the vector hybridization signal with plasmid DNA of a known dilution concentration that was blotted onto the same filter. The radioactivity on the hybridization filter and the optical density on the X-ray film were quantified using the Ambis Image Acquisition and Analysis system.
2.3 Protein Analysis To analyze AAV Rep and Cap protein expression, plasmid transfection was performed as described in Section 2.2. Forty-eight hours after transfection, the culture was scraped into cold PBS/Mg (PBS containing 5 mM MgCl2) and harvested by low-speed centrifugation of the cells. The cell pellet was lysed on ice with 200 ml STM-NP buffer (25 mM sucrose, 10 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 0.5% NP-40, 1.0 mM PMSF, 0.1 Mm DTT). The lysate was centrifuged at 2000 Ìg for 5 minutes to obtain a nuclear precipitate. The nuclear pellet was resuspended in 200 ml IPP buffer, ice bathed for 45 minutes and vortexed frequently. Nuclear extracts were centrifuged at 14 000 Ìg to remove DNA and nuclear debris. The supernatant was mixed with SDS-PAGE sample buffer, heated at 100 ° C for 5 minutes, and separated on a 14 mA 10% SDS-PAGE gel overnight. The protein was transferred to a nitrocellulose membrane using a Trans-Blet SD semi-dry transfer tank (Bi-Rad). The filter was blocked in 2% milk powder dissolved in PBS for 1-2 h, followed by rabbit anti-Rep (Trempe et al., 1987) or hamster anti-Cap (American Type Culture Collection) primary antibody (200-fold diluted with PBS containing 1% milk powder) at room temperature. Incubate for 2 hours or 4 ° C overnight. The blots were washed 3 times with PBS containing 0.05% Tween-20 for 10 minutes each time. A suitable alkaline phosphatase-conjugated secondary antibody was diluted 2000-fold with PBS containing 1% milk powder and incubated for 1 hour at room temperature. The cells were washed 3 times with PBS-Tween-20 buffer, and blotted with NBT and BCIP dissolved in dimethylformamide for 5-20 minutes.
2.4 DNA analysis For the analysis of viral DNA, packaging transfection was performed as described in Section 2.2. Forty-eight hours after transfection, the culture was scraped into cold PBS/Mg and harvested by low speed centrifugation. Extrachromosomal DNA was isolated from the outside of the cell by the method of Hirt (Hirt, 1967). Briefly, the cell pellet was resuspended in 200 ml of lysate (10 mM Tris, pH 8.0, 1.0 mM EDTA, 1.0% SDS) and 40 mg of proteinase K was heated. After 2 hours of digestion at 37 ° C, 50 ml of 50 M NaCl was added and ice bathed for 4 h. The solution was centrifuged at 14000 Ìg for 30 minutes at 4 ° C to remove the chromosome deposit. The supernatant was treated with 5 ml of RNase mixture (Ambion, 5 mg RNase A, 100 units of RNaseT1) at 37 ° C for 2 hours, extracted once with phenol/chloroform (1:1), and extracted once with chloroform. The DNA was precipitated with 2 volumes of ethanol and treated with DpnI to remove the input plasmid DNA. The DNA was separated on an agarose gel, transferred to a nitrocellulose membrane (Hirt, 1967) as described above, and UV-crosslinked to the filter using Stratalinker (Stratagene). The filter was hybridized with a gpp gene-specific 32P probe or an AAV-specific probe. Hybridization and washing were performed as previously described. Hybridization probes were prepared using a random labeling kit. The film was exposed to X-ray film. To estimate the amount of rcAAV, 1 Ì106 293 cells were infected with a dilution of Ad(moi) and CsCl2 purified wild-type AAV or vAVgal. The vAVgfp stock prepared by transfection with pAAV/Ad or pSH3/5 as a helper plasmid was used to transduce Ad-infected cells. After 48 hours, the culture was harvested and viral DNA was prepared and analyzed by agarose gel electrophoresis and Southern hybridization.
3. Results
3.1 The most common protocol for the preparation of recombinant AAV vectors using rAAV packaging of AAV/Ad helper plasmids involves co-transfection of Ad infection with AAV-based vector plasmids containing the gene of interest, viral TR elements, and helper plasmids providing Rep and Cap proteins. Tissue culture cells. One of the most commonly used helper plasmids is pAAV/Ad (Samulski et al., 1989). The main disadvantage of this method is the contamination of Ad in the carrier stock, the generation of replicable AAV, and the inefficiency of the entire process. To overcome these problems, we constructed a novel helper plasmid containing the AAV rep and cap genes and Ad E2a, E4 and VA. When the plasmid was used in combination with a highly transfectable 293 cell line (Graham et al., 1977), it contained the Ad E1a and E1b genes, and all of the components required for packaging the rAAV vector were present. To test whether these plasmids can package an AAV-based vector, we co-transfected 293 cells with pTR-UF5 (Peel et al., 1997) expressing a green fluorescent protein and a helper plasmid (Fig 1A) using lipofectamine. method. To determine the optimal ratio of the two plasmids we used several different ratios. As a control, we also used Ad-5 (15 m. oi) to infect 293 cells, and then transfected them with the vector plasmid and pAAV/Ad as a helper plasmid. After 72 hours, the cells were harvested according to the method of Section 2.2, and lysed for transduction. Lysates were used in triplicate to transduce Ad-infected HeLa cells. Green fluorescent cells were counted by fluorescence microscopy after 3 days. The results of several experiments are listed in Table 1. It is clear that in these experiments comparing packaging efficiency with the pAAV/Ad helper plasmid, we obtained more rAAV using the pSH3 and pSH5 plasmids. We routinely obtained more rAAVs with helper plasmids than pAAV/Ad and Ad infections. The highest yield per cell was 135 T.U. 80 times higher than that obtained with the pAAV/Ad control. A larger proportion of the other vector plasmids than the helper plasmid did not produce a large amount of rAAV. Subsequent genomic copy number determined by dot blot hybridization revealed that the particle-infection ratio was approximately 100 for Ad-infected Hela cells for the vector expressing gfp. The highest yield obtained in these experiments was approximately 1.36 Ì104 DNase resistance genome per cell. These experiments demonstrate that the pSH3 and pSH5 plasmids fully support the efficient rAAV packaging without Ad co-transfection.
3.2. Rep and Cap Protein Expression To demonstrate that the helper plasmid is capable of expressing AAV protein, 293 cells were co-transfected with TR-UF5 and a different helper plasmid as described in Section 2.2. These cultures were harvested 48 hours later, harvested, lysed and analyzed by SDS-PAGE and immunoblotting. Analysis of Rep protein expression by Rep-specific antibodies (Fig. 2A) revealed that pAAV/Ad (lines 4 and 5) were significantly lower than pSH3 (both 6 and 7) or pSH5 (lanes 8 and 9) (1:3 or Protein level of 1:5 ratio). In Fig. 2A, we only point out the location of Rep78 and Rep52, since Rep68 co-migrates with a cross-reactive protein (as seen under Rep78) and the level of Rep40 is too low to be detected in nuclear extracts. Cap expression was analyzed using Cap-specific antibodies (Fig. 2 shows that when pSH3 and pSH5 helper plasmids were used, the Cap expression was significantly increased compared to pAAV/Ad. As expected, in pTR-UF5 (lane 2) and pCDMRep (p. There was no capsid expression in the transfected cultures. Higher levels of Cap protein can be explained by the fact that the yield of rAAV produced by pSH3 and pSH5 is significantly higher than that of pAAV/Ad. Others indicate that the yield of Cap expression level for rAAV is Limited (Vincent et al, 1997; Li et al, 1997). Although the level of Rep78 from pSH3 and pSH5 was significantly higher than that from pAAV/Ad, we did not find a decrease in vector yield. Overexpression of Rep78 indicates production of rAAV There are limitations (Li et al, 1997; Vincent et al, 1997). In addition, there is no significant difference in AAV protein expression between pSH3 and pSH5.
3.3. Viral DNA Analysis To analyze the amount and type of vector produced by these new packaging plasmids, we co-transfected the pTR-UF5 plasmid and the different helper plasmids as described in Section 2.2. After 48 hours of transfection, cells were harvested and extrachromosomal DNA was isolated. Low molecular weight DNA is digested with DpnI (degrading unreplicated plasmid DNA). The digested DNA was subjected to agarose gel electrophoresis and a gfp-specific probe (Fig. 3A) or an AAV-specific probe (Fig. 3 for Southern hybridization. AAVRFM and RFD were found in all DNA samples containing Rep protein. (Fig. 3A: Lane 2-8). The level of DNA of the replicated vector is pAAV/Ad (Fig. 3A, 3 and 4) pSH3 or pSH5 (Fig. 3A, pSH3 5 and 6 lanes, pSH4 7 and 8 lanes) It is almost identical when used as a helper plasmid. A similar membrane is probed for replication-producing AAV, and when pAAV/Ad is used (Fig. 3B, lanes 3 and 4), the replicated DNA is found, but When pSH3 and pSH5 were used as helper plasmids, they were not found (Fig. 3B, 5 and 6). These results indicate that the pSH3/5 helper plasmid fully supports AAV vector DNA replication, but rcAAV produced compared to pAAV/Ad helper plasmid. Less 3.4. Analysis of AAV with replication ability Although there is no significant homology between pAAV/Ad and pTR-UF5, replication competent AAV is able to pass the vector plasmid and the rep and cap genes in pAAV/Ad. Recombination between the ends (Allen et al, 1997; Wang et al, 1998). To assess the level of rcAAV produced by co-transfection of the helper and vector plasmids, sense of AD The 293 cells were infected with the wild type AAV or rAAV-gfp vector of increasing moi. After 2 days of transduction, the low molecular weight viral DNA was isolated and analyzed by Southern blotting with radiolabeled AAV probe. When 106 293 cells were infected with moi 10 AAV DNA was found in the -3 (approximately 104 genome) wild-type virus (Fig. 4, Lane 2). When the cells were transduced with rAAV-gfp prepared with the pAAV/Ad packaging plasmid, the replication-capable AAV was also found. (Fig. 4, 7). After passage of Ad-infected cells, the number of rcAAVs from a pAAV/Ad packaging experiment was between 104 and 105 particles (determined by the number of copies of the genome). However, in 4 different No replication-producing AAV was found in the preparation of small-scale rAAV-gfp (Fig. 4, 8-11). Using the same analytical method, large-scale preparation of rAAV b-gal vector from 20 Ì375px culture dish was not found. rcAAV (Fig 4 12-17). From the analysis of the largest amount of gfp vector (8.0 Ì108 particles) and the lowest amount of wild-type AAV (104 particles) in these gels, we estimated that the amount of rcAAV contamination was less than 8.0 Ì104 The carrier particles are 0.00125% of the concentration of a genome or vector. Subsequent small and large gauges In the mold carrier preparation experiment, rcAAV contamination was analyzed after 2 passages in Ad-infected 293 cells, and no contamination was detected.
4. discuss
Gene therapy vectors obtained from AAV are being widely used in various acquired diseases and genetic diseases. Proliferation of the AAV vector is hampered by the labor-intensive methods required to generate the vector. Here, we describe the construction and analysis of a novel helper plasmid that allows for the production of no Ad vector and significantly increases the yield of recombinant vectors. The helper plasmid contains the E4, E2a and VA RNA genes of Ad and the AAV rep and cap genes. When introduced into Ad E1a/E1b transformed 293 cells, all of the desired Ad helper genes were provided, enabling efficient vector amplification and packaging. A similar approach has been reported in which the helper gene of Ad is provided by plasmid transfection rather than viral infection (Grimm et al, 1998; Matsu***a et al, 1998; Xiao et al, 1998b). There is no contamination at all and the purification of the vector is simplified. Grimm et al. (1998) describe a procedure similar to the construction of our Ad helper plasmid and used for packaging. An increase in vector yield was obtained with this new helper plasmid. We have obtained transduction units that exceed 100 gfp expression vectors per transfected cell. Dot hybridization analysis revealed that this corresponds to the yield of more than 104 vector genomes per cell. We believe that the vector packaging system for the improved pSH3/5 helper plasmid from pAAV/Ad plasmid is due to (1) higher levels of AAV protein expression (Fig. 2) and (2) introduction of only two reagents (vector and helper plasmid) Instead of 3 reagents (Ad infection, vector and helper plasmid) into the cultured cells. Our new plasmid expresses more Cap protein than pAAV/Ad, which may fully explain the increase in vector yield. It has been reported that high levels of Rep78 expression inhibit vector production (Li et al, 1997; Vincent et al, 1997). However, the level of Rep78 accumulation from pSH3/5 was higher in the experiment than from pAAV/Ad, but the yield from pSH3/5 was higher. It is apparent that increased Cap expression from pSH3/5 is sufficient to overcome the inhibition of Rep78. Early AAV studies estimated that each infected cell produced 105-106 genome and 104-105 tissue culture infected units (Parks et al., 1967; Rose and Koczot, 1972). There is therefore the potential to further improve this carrier system. One modification that may increase vector production is to replace the ATG promoter codon with ACG to reduce the expression of Rep, which can effectively increase vector production (Li et al., 1997). Another limitation of AAV packaging systems that plague carrier generation is the generation of rcAAV. Although there is no large-scale (>8 bp) homology between most AAV vectors and helper plasmids, rcAAV is still produced by non-homologous recombination (Allen et al, 1997; Wang et al, 1998). Our helper plasmid contains the same cap and rep genes of AAV as the wild type virus. Thus, it is possible to generate rcAAV due to non-homologous recombination. However, no analysis of our small-scale and large-scale preparations found any rcAAV. The experiment shown in Fig. 4 is the result obtained by passage of Ad infected Hela cells once. Subsequently, the experimentally prepared vector was found to have no rcAAV in 2 passages in the Ad-infected cells. The absence of rcAAV is a significant advantage of this system because the virus expressed by the rep gene can affect transduction in vivo (Halbert et al., 1997). The helper plasmid we developed includes improvements to some previously published packaging systems. We excluded the inverted terminal repeat of Ad, which showed that rcAAV was produced when the AA vector was packaged with the pAAV/Ad helper plasmid (Wang et al., 1998). By assembling the AAV rep and cap genes into a plasmid that also contains the Ad helper gene, we do not need to have a 3 plasmid as in other systems (Matsu***a et al., 1998; Xiao et al., 1998b; Grimm et al., 1998). Transfection system. Grimm et al. (1998) developed a similar system to carry AAV and Ad early genes on the same plasmid and produced similar levels of vector. In summary, we developed an improved plasmid transfection system to generate recombinant AAV vectors. The simplicity of vector generation, increased vector yield, and lack of rcAAV or Ad contamination make this system particularly useful for preclinical screening of multiple vector constructs.
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