Overcome The Difficulties of Bioanalysis in the Application of Gene Therapy Drugs with AAV as a Vector

Preface

The concept of gene therapy was proposed in the early 1970s. The original concept was to introduce a normal copy of a gene to replace a mutated counterpart. Although there are now more alternatives, such as gene editing and base editing, it is still one of the main strategies that Accurant Biotech Inc., applies.

Gene therapy can be given through “ex vivo” or “in vivo” methods. “Ex vivo” gene therapy involves isolating cells from the patient, modifying and expanding "in vitro", and then infusing them back into the body to achieve the therapeutic effect, such as CAR-T-cell therapy. Within “Vivo therapy”, new genes are inserted directly into the lesion site of the patient through the viral vector. Theoretically, “in vivo” gene therapy can achieve the genetic modification of any kind of cells. It is no longer limited by the types of cells like ex vivo gene therapy does, which is the biggest advantage of "in vivo" gene therapy over "ex vivo" gene therapy. But it is still technically difficult to modify genes of all kinds of cell types, such as AAV virus vector gene therapy. In addition, "in vivo" gene therapy also eliminates the tedious steps of cell collection, gene modification, and cell expansion in the "ex vivo" method. Gene therapy drugs with AAV as the vector use a variety of bioanalytical methods in the CMC stage, preclinical and clinical stages of the product. Different technology platforms provide different solutions for gene therapy drugs using AAV as a vector.

Analysis Difficulty 1: AAV-based gene therapy products need to examine a variety of quality attributes in the CMC stage, including identification, content/potency, purity, impurities, and safety. Besides, content and potency assessment are the biggest difficulties. Accurant Biotech has extensive experience in genome titers and infectious experiments. Particularly for the viral titers, quantitative detection schemes for AAV genomic copy number titers of multiple serotypes have been developed not only to determine the titer of the virus but to investigate viral integrity as well.

Accurant Biotech used Naica digital PCR technology to optimize the quantification methods for the copies of three different loci of ITR (CY5 markers), the gene of interest (GOI, HEX labeling), and transcriptional regulatory element WPRE (FAM labeling) in recombinant adeno-associated virus (rAAV) and virus genome. Accurant Biotech investigated and compared the conditions of the pretreatment methods of different viral samples during data analysis. It is found that the linearization of ITR hairpin structure and the cleavage condition of capsid protein in the pretreatment of viral samples were particularly important for the accuracy of viral genome copy number quantification. It provides a basis for the establishment of the standard digital PCR detection methods in the quality control system of the gene therapy viral vectors.

Fig 1, The 1D scatter plot of cdPCR detection of three targets WPRE/GOI/ITR

Figure A shows a 1D scatter plot for WPRE detection, figure B shows a 1D scatter plot for GOI detection, figure C shows a 1D scatter plot for ITR detection before Msp I digestion, and figure D shows 1D scatter plot of ITR detection after MspI digestion. Blue stands for WPRE positive droplet group, green stands for EGFP positive droplet group, red stands for ITR positive droplet group, and gray stands for negative droplet group. When the ratio of WPRE or GOI to ITR is closer to 1:2, the genome of the product is more intact.

Analysis Difficulty 2: The preclinical tissue distribution and clinical shedding studies of AAV gene therapy. Since the drug enters the body and then various organs, according to the mechanism of action and the purpose of treatment, the preclinical study of tissue distribution can predict the drug safety and guide the clinical protocol. Shedding analysis at the clinical stage will be determined by analysis of excreta after drug administration and this usually includes fluorescence PCR reactions and infectivity tests.

Accurant Biotech has experience in preclinical tissue distribution and clinical phase vector shedding of various gene therapy drugs, including oncolytic virus and different serotype AAV gene therapy drugs. Both assays using fluorescence quantitative PCR require the synthesis of primers and probes with strong specificity and sensitivity. During the DNA extraction of various tissue samples and metabolite samples, some PCR inhibitors may be introduced, and the difference in the amount of product during the extraction process needs to be considered.

Fig 2, Flowchart of Accurant Biotech Conducting PK/Shedding Research

Analysis Difficulty 3: Immunogenicity is an important factor affecting the safety and efficacy of AAV gene therapy products. AAV gene therapy not only needs to consider humoral immunity but also cellular immunity in the evaluation of immunogenicity. Humoral immunoassays may include detection of anti-capsid protein antibodies/neutralizing antibodies and anti-expression product antibodies. The LBA or cell-based assay is commonly used for this analysis. Cellular immunoassays involve analysis of T cell responses against capsid proteins or T cell responses against expression product antibodies. Accurant Biotech has the methodology to develop different strategies and detection equipment for humoral immunity or cellular immunity. Meanwhile, a well-developed method can be used to carry out preclinical immunogenicity assessment after the AAV capsid protein is modified.

Using the ELISpot platform, Accurant Biotech has developed and validated a methodology based on ELISpot technology to detect a few immune cells secreting IFN-γ after stimulation of human P BMC by CEF. It leads to the establishment of a relatively complete methodological development and verification system. At the same time, methodological development is being carried out on the number of immune cells secreting INF-γ after stimulation of PBMC by different serotypes of AAV. Moreover, specific method development and validation are being designed based on the differences of various serotypes of AAV vectors.

Fig 3, the application of Accurant Biotech in ELISPOT cell immunity

1. Select appropriate stimuli and immune cells (PBMC, B cells, T cells) and add to the ELISpot plate.
2. Incubate and activate immune cells secrete cytokines.
3. ELISpot plates coated with anti-cytokine antibodies capture cytokines secreted by activated immune cells.
4. Wash plate to remove the cells, add the secondary antibody reaction, enzyme-linked detection, and the activated immune cells form a regular round shape spot.

Accurant Biotech has different detection and technology platforms. Starting from mid-2022, Accurant Biotech built a second-generation sequencing platform, including a well-designed, well-equipped, and independent high-throughput sequencing laboratory. The laboratory design follows the specific detection processes of high-throughput sequencing and the principles of pollution prevention. The experiment consists of a dedicated corridor with zoning designs for reagent preparation, specimen preparation, amplification area I, hybridization capture/amplification area II, sequencing, and electrophoresis. The laboratory is fully equipped and verified. The current laboratory instruments include TC-96/G/H(b)B (gene amplification instrument), QuantStudio 5 Real-Time PCR Instrument (QuantStudioTM 5 real-time quantitative PCR instrument), Agilent 4150 (segment analyzer), Covaris M220 (nucleic acid sonicator), E-Gel Power Snap (gel electrophoresis system), Qubit (fluorometer), NanoPhotometer N60 Touch (ultra-micro spectrophotometer), etc. They fully meet the needs of high-throughput sequencing services. It also provides a better service for the increasing demand for AAV gene therapy drugs in the CMC, preclinical, and clinical stages.