Services and Pricing

The expected yields shown below are based on the average yields of reporter gene vectors generated for AAV serotypes 1, 5, 7, 8, 9 and rh10 with AAV serotypes 2 and 6 having somewhat lower yields. Although we try our best to achieve the same yields for custom vector preparations as we do for our reporter gene vectors, our titer release criterion for custom vectors is ½ of the anticipated yield of our reporter gene preparations. If we do not meet the titer release criterion we have established for our vectors, we will repeat the preparation once again at no additional charge to the investigator who will be provided with the preparation that resulted in the highest yield. Our endotoxin release criterion for vector lots to be used in animal studies is < 5 endotoxin units per mL. There is a $159 charge assessed for shipping material for each shipment.

Our release criteria do not apply to AAV vectors with the following:

• Oversize genomes (>4.9 kb from ITR-ITR including ITRs)
• Self-complementary or double-stranded genomes
• Genomes expressing shRNA under Pol II or Pol III promoters
• Genomes expressing toxic genes under ubiquitous promoters

Volume: 0.75 to 1 ml

Plasmid needs: 0.3 mg for transgene

Typical vector yields: 5e12 to 1e13 genome copies

Services included: ddPCR titration and Endotoxin assay

Volume: 1.7 to 2 ml

Plasmid needs: 0.5 mg for transgene

Typical vector yields: 1e13 to 3e13 genome copies

Services included: ddPCR titration and Endotoxin assay

Volume: 2.5 to 3 ml

Plasmid needs: 0.75 mg for transgene

Typical vector yields: 3e13 to 6e13 genome copies

Services included: ddPCR titration, Endotoxin assay, Purity assay

Volume: 4.5 to 6 ml

Plasmid needs: 1 mg for transgene

Typical vector yields: 5e13 to 1e14 genome copies

Services included: ddPCR titration, Endotoxin assay, Purity assay

An integral part of the Penn Vector Core is its robust quality control (QC) program which is carried out by a separate QC group. The QC assays have been developed and optimized for each vector, and criteria are in place which must be met before the release of any vector from our facility. Standard operating procedures are followed and batch record forms are utilized for each assay conducted at the Penn Vector Core.

The outcome of gene therapy research can be dramatically influenced by the quality of the viral vector. The QC group provides numerous services to assess the quantity and quality of AAV vectors produced by the Penn Vector Core. The mission of the QC group is to provide accurate information on the quality of viral vectors provided by the Penn Vector Core and to continually strive to improve the accuracy and scope of services offered by developing and optimizing novel assays useful for assessing the efficacy of gene therapy vectors.

For researchers looking to outsource the cloning of plasmid for AAV vector production, Penn Vector Core offers cloning services to make your transgene cassette. We can use template DNA or help you to place a gene synthesis order through a 3rd party vendor. We will also need to include Plasmid Amplification and Characterization services to ensure that we have the correct transgene cassette and have high-quality plasmid for optimal production of your vector.

Don't have enough plasmid to send for vector production? Penn Vector Core can amplify your plasmids for you and characterize the DNA to ensure its identity before AAV production. Full plasmid characterization includes determination of concentration, assessment of A260/280 ratio, detection of endotoxin, and plasmid identity by restriction enzyme digestion and Next Generation Sequencing. For transgene plasmids, we sequence to verify that no mutational changes have occurred in any DNA that will be packaged into the AAV capsid. For AAV capsid plasmids, the presence of the correct cap gene is verified by full-length sequencing.

Penn Vector Core strives to make the purest vector possible. We can test the purity of a vector preparation by SDS-polyacrylamide gel electrophoresis to assess host cell protein contamination. Bands corresponding to the viral structural proteins VP1, VP2, and VP3 can be visualized by SYPRO Ruby staining, and their size and relative intensity assessed with respect to contaminating proteins.

Contaminating endotoxin in plasmid preparations can affect transfection efficiency, and endotoxin contamination in vector preparations can alter the immunogenic properties of the final product. The endotoxin assay is carried out using the Limulus Amebocyte Lysate (LAL) gel-clot method (QCL-1000, Bio Whittaker) to detect and quantitatively determine the gram-negative bacterial endotoxin level in the plasmid or vector preparations. Our release criterion for vector lots to be used in animal studies is < 5 endotoxin units per mL.

The accuracy and reliability of AAV titration are paramount for all work involving AAV vectors. For this reason, the QC group has put extensive effort into ensuring that the genome titrations are conducted with precision and consistency. A number of standards, validation samples (both viral and plasmid), and controls (for background and DNA contamination) have been introduced into the PCR titration assay. The QC group maintains stocks of primers and probes designed for fast and reproducible AAV vector titration.

In order to improve sensitivity, reproducibility and turn-around time, an infectivity assay for AAV vectors has been developed and used as an alternative to standard techniques such as the infectious center assay (ICA). The Taqman TCID50 assay is based upon limiting dilution of the vector and a 50% endpoint determination of viral DNA replication using real-time PCR for sensitive, quantitative calling of positive wells. AAV vectors are serially diluted and a cell line expressing AAV rep and cap is co-infected with these dilutions plus wildtype Ad5 in a 96-well plate format (8 replicate wells per dilution). The presence of AAV rep and adenovirus helper genes allows for the replication of AAV DNA. After a suitable incubation period, DNA is extracted and a 50% endpoint determination is performed by a basic computer program based upon Karbers formula. With a validation sample included in every run, the assay has proven to be accurate and reproducible. A GC: infectivity (GC:I) ratio is calculated based upon the results of both the GC copy number titration and TCID50 assay results. The GC:I ratio is used as a measure of infectivity of the preparation with low GC:I ratios indicating more infectious vector lots. The GC:I ratios between different lots of the same serotype are useful in assessing the relative potency of a particular preparation. However, the infectivity of a vector based on GC:I ratio does not predict the in vivo performance, particularly when comparing different AAV serotypes.