Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5943_Библиотеки_им_академика_М_И_Перельмана

fragments are then end‐repaired and ligated with sequencing adaptors that include sample specific barcodes and P5 and P7 universal Illumina sequencing adaptors. The ligation products are then subjected to PCR to generate sufficient material for the capture reaction. The library of amplified genomic fragments is hybridized with biotinylated RNA or DNA probes tiling across the candidate edited site and flanking sequences. The hybridization products are then purified using streptavidin beads, amplified, purified, and sequenced. This hybrid capture approach is compatible with UMI tagging, which can address artifacts arising from PCR and sequencing errors. The disadvantages of hybrid capture approaches are requirements for substantial assay optimization, including gDNA sheering conditions and PCR conditions. In addition, DNA capture is less efficient for GC‐ and AT‐rich sequences. Since off‐target sites are often located in intronic and intergenic regions the efficient probe development to accommodate all edited sites can be challenging.

decrease in background (false‐positive indel signal) was seen while retaining >98% indels in treated samples [57]. The optimal sequence “window” can be established by careful analysis of oligo incorporation using unbiased genome‐ wide sequencing methods e.g. GUIDE‐seq. The exact genomic position of incor­porated oligo can be used to establish the correct sequence “window” to analyze and quantify indels.

run. The occurrence of sequencing artifacts was decreased by reducing the cluster density on the flow cell during the sequencing reaction and precisely defining the sequence “window” where editing is expected to occur. This method is amenable for invitro studies where multiple biological replicates of treated and untreated samples can be run.

The FDA guidance for bioanalytical method validation[64] underlines the impor­tance of analytical method characterization supporting regulatory submissions. This includes bioanalytical parameters, such as sensitivity, specificity, accuracy, precision, and description of the reference materials. There is currently no spe­cific FDA guidance for analytical method characterization for off‐target detection. This section will focus on our case study of indel quantification in ZFN‐edited hematopoietic stem and progenitor cells using NGS, specifically, how to generate material for making quality controls (QCs) . Reference standards and QCs are important reagents for quantification of most detection methodologies. QCs are commonly used to develop and characterize the assay and to monitor run‐to‐run variability when drug product or post‐infusion patient samples are analyzed. There are several approaches for QC creation that have been explored in the field. One approach is to use engineered cell lines by introducing edits at the same genomic locations as expected in study samples (cell therapy drug product as well as animal and human samples post‐infusion). Such cell lines can be generated by transfecting immortalized cells with studied engineered nucleases to cause a high level of indels, followed by the propagation and banking of these cell lines. The level of indels can be determined by NGS and, ideally, by an orthogonal methodology e.g. ddPCR. QC samples containing different levels of indels then can be gener­ated by mixing a cell line with a known frequency of editing together with WT cells at different ratios[25] or isolated gDNA.

made as needed using the same stock material. Though synthetic DNA can be implemented faster than engineered cells, this approach suffers from its artificial nature. In cell or tissue study samples, indel sequences at a known editing site typically are heterogenous in nucleotide sequence and length. Usually, only a few indel sequences are mimicked by the synthetic DNA, while others are not repre­sented. In addition, synthetic DNA is usually short and therefore an easier target for PCR amplification when compared to the more complex gDNA matrix present in study samples. Some DNA sequences might be difficult to synthesize due to their sequence composition (homopolymers, stem‐loop structures, etc.) which may result in a low yield and high level of sequence errors in synthetic DNA. This can be a common issue for off‐target sites that are often found in non‐coding regions with sequence compositions that may be difficult to synthesize. When synthetic DNA is ordered, it should be carefully evaluated how much material is required to support testing for the entire clinical trial to avoid batch effects.

Therefore, the engineered control cell line approach may be better suited when establishing method feasibility and accuracy if timeline permits. A synthetic DNA approach can be implemented at the sample testing stage with the purpose of assessing run‐to‐run variability.

Droplet Digital™ PCR (ddPCR™) is a technique that allows for absolute quantita­tion of target DNA in complex biological matrices. The PCR reaction is fraction­ated into 20,000nanoliter‐size droplets. As a result of partitioning, target DNA molecules get distributed across the droplets so that each droplet contains zero, one, or a few copies of DNA templates. PCR amplification is then carried out to the plateau phase and the droplets are counted as positive and negative reactions by a droplet reader. The quantity of target DNA can be calculated using the frac­tion of positive droplets and Poisson statistics[65, 66]. This methodology was suc­cessfully adopted to assess NHEJ‐derived indels and implemented in various research studies (GEF‐dPCR – gene‐editing frequencies digital PCR or DSB‐ ddPCR–DSB ddPCR)[67–69] (Figure14.5B). These techniques utilize two differ­ent TaqMan probes carrying two different fluorophores (FAM and HEX) targeting the same PCR product and simultaneously detecting wild‐type and edited alleles.