Cell And Gene Therapy: Research, Development & Regulatory Guidance

Droplet digital PCR  ( ddPCR ) : A third generation of quantitative PCR, water and oil emulsions is used to segment the samples and create droplets to identify and quantify the genetic material in the sample. It is a new PCR technique that directly quantifies DNA copies with an unparalleled degree of precision and without the need for a standard curve or for a high degree of amplification efficiency; all properties that lend themselves to the accurate quantification of both single-stranded and self-complementary AAV genomes. The advantage of ddPCR for the nucleic acid quantification includes:  Unparalleled precision:  the massive sample partitioning afforded by ddPCR enables small fold decrease in target DNA sequences between samples to be reliably measured.   Increased signal-to-noise:  enrich for rare targets by reducing competition that comes from high-copy templates.  Removal of PCR efficiency bias:  error rates are reduced by removing the amp...

  Analytical Consideration For Supporting Biodistribution Studies For AAV Gene Therapy The biodistribution studies should be designed to answer the following questions Profiling and quantity of the DNA/RNA of the gene therapy products in the different tissues Expression and profiling of the target RNA gene or protein products in different tissues Profiling and quantity of the DNA/RNA of the gene therapy products in the different tissues Profiling and quantification of the DNA/RNA of the gene therapy products and biofluids are the primary object of the BD studies. These data provides the selective tropism of the AAV WT, serotypes and AAV variants to the different tissues. It has become even more important as there are increased efforts to identify novel AAVs with improved selective tropism and low immunogenicity. Both screening of natural variants and capsid engineering are extensively employed.   Currently, real-time quantitative polymerase chain reaction (qPCR) is consid...

  Studying Multiple Versions of a Cellular or Gene Therapy Product in an Early-Phase Clinical Trial; Draft Guidance for Industry 11/2022 Human Gene Therapy for Neurodegenerative Diseases; Guidance for Industry 10/2022 Considerations for the Development of Chimeric Antigen Receptor (CAR) T Cell Products; Draft Guidance for Industry 3/2022 Human Gene Therapy Products Incorporating Human Genome Editing; Draft Guidance for Industry 3/2022 Interpreting Sameness of Gene Therapy Products Under the Orphan Drug Regulations; Guidance for Industry 9/2021 Manufacturing Considerations for Licensed and Investigational Cellular and Gene Therapy Products During COVID-19 Public Health Emergency; Guidance for Industry 1/2021 Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs); Guidance for Industry 1/2020 Long Term Follow-up After Administration of Human Gene Therapy Products; Guidance for Industry 1/2020 Testing of Retroviral V...

What is Biodistribution? Biodistribution (BD) is the in vivo distribution, persistence, and clearance of a gene therapy (GT) product at the site of administration and in target and non-target tissues, including biofluids (e.g., blood, cerebrospinal fluid, vitreous fluid), in biologically relevant animal species. Nonclinical BD studies entail the use of analytical methods to detect the GT product and transferred genetic material in collected samples and can include methods to detect the expression product of the transferred genetic material. Characterization of the BD profile following administration of a GT product in animals is a critical component of a nonclinical development program. BD characterization data are critical in: overall interpretation of the study findings to enable a better understanding of the relationship of various findings (desired and undesired) to the administered GT product.   ascertaining a potential benefit: risk profile of the GT product before admin...

Step Loop RT-PCR for Detection of Small Interfering RNA (siRNA) The recent publications described a novel used the novel method for the detection of siRNAs using a TaqMan®-based approach. This approach utilizes similar strategy that has been used for microRNA detection. The approach is illustrated in below.  In brief, the RT step occurs in the presence of a stem-loop RT primer that is complementary to the last 6–10 bases of the 3′ end of the antisense strand of the target siRNA. The stem-loop primer contains an additional universal sequence at the 5′ end that facilitates a TaqMan-based detection strategy in the subsequent qPCR step. As in the case of microRNA, the forward primer for qPCR is sequence-specific for the target siRNA. For sequence compositions that yield a low predicted melting temperature (Tm), the forward primer is designed as a tailed primer to help increase Tm. Stem Loop PCR for SiRNA Detection Step 1: Preparation of liver and plasma samples for the quanti...

Adeno-associated virus (AAV)-derived vectors are among the most promising gene therapy tools because of their safety and ability to mediate long-term transgene expression. The quantification of viral nucleic acid by quantitative polymerase chain reaction are widely employed to study the biodistribution of AAV products. The procedure by Betroff et al is described below as an example. Collection and Preparation of Animal Tissues: Homogenize the frozen animal organs by applying mechanical force using liquid nitrogen and a pestle to obtain a fine dry powder.  Extraction of Genomic DNA extraction  Perform the extraction of DNA using using the protocol of the DNeasy Blood & Tissue Kit (“Purification of Total DNA from Animal Tissues (Spin-Column Protocol)” in the DNeasy Blood & Tissue Handbook; QIAGEN, Hilden, Germany). Add 20 µl Proteinase K to the tissue homogenate. Mix thoroughly by vortexing, and incubate at 56°C until the tissue is completely lysed.  Vortex occasio...