Cell And Gene Therapy: Research, Development & Regulatory Guidance

Adeno-associated virus (AAV) is a popular vector for gene therapy due to its low immunogenicity and ability to deliver therapeutic genes to target cells. However, recent studies have shown that some individuals may develop a T cell response against AAV capsid proteins, which can limit the effectiveness of AAV-based gene therapies. Wang et al. (2019) found that up to 70% of participants in AAV-based gene therapy clinical trials had circulating CD8+ T cells that recognized specific AAV capsid proteins and were capable of eliminating AAV-transduced cells in vitro. The authors also found that the presence of preexisting AAV capsid-specific CD8+ T cells was associated with reduced transgene expression and therapeutic efficacy in some participants. Mingozzi et al. (2013) investigated CD8+ T cell responses to AAV capsid in healthy human subjects and found that the majority of participants had detectable AAV capsid-specific CD8+ T cell responses. However, these responses did not affect transge...

The ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) provides guidance on the conduct of nonclinical biodistribution studies for AAV gene therapy products. The guidance is designed to ensure that biodistribution studies are conducted in a manner that is consistent with regulatory requirements and that provides relevant information on the distribution and persistence of the AAV vector in different tissues and organs. According to the ICH guidance, nonclinical biodistribution studies for AAV gene therapy products should include the following: Dose selection: The highest dose tested in the study should be the intended clinical dose, or a dose that is expected to produce a similar level of transgene expression. Study design: The study should include a minimum of three animal species, with at least two non-rodent species. Sample collection: Samples should be collected at multiple time points after administration of the AAV vector, wit...

The choice of method for assessing AAV gene integration will depend on a variety of factors, including the sensitivity and specificity of the method, the resources available, and the research question being addressed. There are several methods for assessing AAV gene integration in host tissues. Here are a few examples: PCR-based methods: Polymerase chain reaction (PCR) is a widely used method for detecting AAV gene integration in host tissues. PCR can be used to amplify specific regions of the AAV genome that flank the integration site, allowing for the identification of the site of integration. Inverse PCR: Inverse PCR is a modification of the standard PCR technique that is used to amplify DNA sequences that flank a known region of DNA. This method has been used to identify AAV integration sites in a number of studies. Targeted sequencing: Targeted sequencing is a method that allows for the selective amplification and sequencing of specific regions of the genome. This can be used to i...

Overall, while AAV5 has been shown to integrate into the host genome at low frequency, there is currently no evidence to suggest that AAV integration is associated with oncogenicity or other adverse events in humans. Ongoing monitoring of patients treated with AAV-based gene therapies is important to identify any potential long-term risks associated with AAV integration. Following are the results from the selected publications Nakai, H., et al. (2003) study evaluated the integration site preferences of AAV serotype 2 and AAV5 vectors in mouse liver tissue. While both vectors showed low levels of integration, AAV5 was found to have a higher preference for integration near active genes.  Mingozzi et al. (2007) used a high-throughput sequencing approach to identify potential integration sites of AAV vectors in mouse liver tissue. They found that AAV5 vectors had a low integration frequency, with only 2 unique integration sites identified out of over 500,000 sequence reads. Zincarelli ...

 The circulating drug binds to the anti-drug antibodies (ADA) in the samples and interferes with the detection of ADA. The circulating drug severely reduces the sensitivity of the assay and increases the false-negative rate during clinical bioanalysis.  Drugs can interfere with ADA detection by two mechanisms;  a) Circulating drugs form immune complexes with ADA b) Circulating drugs compete with the detection reagents in the assay  Biotin-Drug Extraction and Acid Dissociation  (BEAD)  procedure: BEAD method has shown to effectively remove the circulating drugs from the serum samples and reduce the drug interference in the assay. The BEAD method utilizes acetic acid to dissociate the drug-ADA complex. The sample is rapidly neutralized in the Tris-HCl containing an excess of biotinylated drug and allow binding to ADA to biotinylated drugs.  The streptavidin-coated magnetic Bead is used to capture the biotinylated drug-ADA complex and remove any free circ...