Exploring Our Latest Publication on Bioanalysis: Review on the Bioanalysis of Non-Virus-Based Gene Therapeutics

In recent years, there has been an increasing number of gene therapeutics approved and entering clinical trials, bringing new hope for the treatment of many inherited and acquired diseases. Compared to conventional treatments that target functional proteins, gene therapy can achieve enduring or curative effects via gene activation, inhibition, and editing. However, delivering DNA/RNA to target cells involves numerous barriers, and the delivery strategies can be divided into viral-based and non-viral-based vectors. Recently, the bioanalysis team of WuXi AppTec DMPK published an article on the Bioanalysis titled “Review on the bioanalysis of non-virus-based gene therapeutics”, published by Taylor & Francis. This article summarizes various bioanalytical strategies for non-virus-based gene therapeutics, as well as the challenges and future perspectives of bioanalysis in this field. This blog will briefly introduce the PK/TK, biodistribution, and immunogenicity evaluation strategies for the components of non-virus-based gene therapeutics, including the gene payload, delivery vector, and expressed proteins.

Bioanalysis of the gene cargo: oligonucleotides

The choice of proper oligonucleotides bioanalysis platform varies depending on the assay requirements, such as sensitivity, ability to discriminate metabolites, multiplex, throughputs, and quantification or semi-quantification. Strategies including low- or high-resolution liquid-chromatography mass-spectrometry (LC/MS), hybridization ligand binding assay (LBA), hybrid LBA-LC/MS, and polymerase chain reaction (PCR) are mainly employed for the oligonucleotides bioanalysis.

Bioanalysis of the gene cargo: long nucleic acid

For longer nucleic acids like mRNA or DNA, the whole sequence often spans thousands of nucleotides. Generally, the branched DNA (bDNA), qPCR, and dPCR-based platforms are primarily used for mRNA/DNA quantification. Because of its extremely high molecular weight and poor ionization efficiency, it is almost impossible to obtain intact quantification of long nucleic acids through LC/MS. However, the LC/MS method could be a powerful tool to characterize mRNA, including the nuclease-based sequence mapping, RNA post-transcriptional modification (PTM) evaluation, and integrity evaluation (poly-A tail and cap).

Bioanalysis of the non-viral-based gene vector

The bioanalysis of non-viral vectors is significantly more challenging than the bioanalysis of conventional small molecular drugs due to the large size, heterogeneity, and complexity of several components. The selection of an appropriate bioanalysis method for measurement often depends on the vector's physical properties and chemical composition in a fit-for-purpose design. Multiple strategies can be used to evaluate PK and biodistribution for the non-viral based gene vector, including the LC/MS, inductively coupled plasma mass spectrometry (ICP)-MS, LBA, radiolabeling, and fluorescence labeling.

Bioanalysis of the expressed protein

For gene therapeutics expressing protein, the bioanalysis strategies are similar to those of protein-based therapeutics. Multiple assay platforms can be used for the PK and biodistribution evaluation of the gene therapeutics expressed protein, including LBA, LC/MS, hybrid LBA-LC/MS, western-blot (WB), and immunohistochemistry (IHC). The choice of proper assay platforms depends on the requirement of assay sensitivity, dynamic range, throughput, and turnaround time. Several mRNA-encoded vaccine antigens, therapeutic antibodies, and protein replacement were determined by ELISA assay.

Immunogenicity evaluation

The immunogenicity evaluation of the non-viral based gene therapeutics shall consider at least three aspects: the immunogenicity against the gene cargo (e.g., again DNA/RNA transgenes), the immunogenicity against the gene vector (e.g., liposome or polymers), and the immunogenicity against the expressed protein (e.g., mRNA/DNA based protein replacement therapy). To be noticed, the pre-existing anti-drug antibody (ADA) may also need to be monitored.

Final thoughts

Bioanalysis plays a crucial role in the development of non-viral-based gene therapeutics. The composition of non-viral gene therapeutics varies, which could significantly impact the bioanalysis assay design. WuXi AppTec DMPK has accumulated extensive experience in preclinical bioanalysis and has undertaken multiple projects in the bioanalysis of non-viral-based gene therapeutics, allowing for rapid establishing and validating of preclinical bioanalytical methods to meet regulatory IND requirements.

Authors: Xue Zhang, Maotian Zhou, Lili Xing

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Committed to accelerating drug discovery and development, we offer a full range of discovery screening, preclinical development, clinical drug metabolism, and pharmacokinetic (DMPK) platforms and services. With research facilities in the United States (New Jersey) and China (Shanghai, Suzhou, Nanjing, and Nantong), 1,000+ scientists, and over fifteen years of experience in Investigational New Drug (IND) application, our DMPK team at WuXi AppTec are serving 1,600+ global clients, and have successfully supported 1,500+ IND applications.