Cell and Gene Therapy Lesson 1: Acronyms, Buzzwords and Crucial Lingo

Heather Myler, Ph.D., director, PPD^™ Laboratory services, bioanalytical lab, and Peter Wunderli, Ph.D., research fellow, PPD Laboratory services, GMP lab, talk about harmonization of language in the field.

Advanced therapy medicinal products (ATMP) are expanding into the biopharmaceutical space with an anticipated growth of 1,000% from 2017 to 2024, according to Evaluate Pharma market reports from January 2019. Numerous technologies and analytical strategies are applied to evaluate the critical quality attributes, safety and efficacy of this modality, with many lacking clear regulatory guidance and industry harmonization. To begin this harmonization, we must all first be speaking the same language. While there are some differences in how regulatory agencies classify these products, the European Medicines Agency (EMA) and U.S. Food and Drug Administration (FDA) have provided some definitions to get us started.

For example, if you were wondering what the difference is between gene and cell therapies, the American Society of Gene and Cell Therapy (ASGCT) has this to say: “Gene therapy involves the transfer of genetic material, usually in a carrier or vector, and the uptake of the gene into the appropriate cells of the body. Cell therapy involves the transfer of cells with the relevant function into the patient,” where the cells also may be (ex vivo) genetically modified.

ATMPs include the following medicinal products for human use: gene therapy medicinal products (GTMP), somatic cell therapy medicinal products (SCTMP) and tissue-engineered medicinal products (TEMP). There are more in-depth descriptions:

• GTMPs contain genes that result in a therapeutic, prophylactic or diagnostic effect. They work by inserting recombinant genes into the body, usually to treat a variety of diseases, including genetic disorders, cancer and long-term diseases.
• SCTMPs contain cells or tissues that have been manipulated to change their biological characteristics, or cells or tissues not intended to be used for the same essential functions in the body. They can be used to cure, diagnose and prevent diseases.
• TEMPs contain cells or tissues that have been modified so they can be used to repair, regenerate or replace human tissue.

Other terminology and definitions relating to ATMPs including the following:

• Antisense oligonucleotides (ASO), small pieces of DNA or RNA that can bind to specific molecules of RNA blocking protein production¹.
• Small interfering/silencing RNA (siRNA, RNAi), double-stranded RNA (20-30 base pairs) that inhibits the expression of genes by triggering degradation of messenger RNAs with complementary sequences.¹
• MicroRNA (miRNA), a short segment of RNA (21 to 25 nucleotides) that suppresses gene expression by binding complementary segments of mRNA, interfering with the formation of proteins during translation.¹
• Aptamer, a short segment of DNA, RNA or peptide that binds to a specific molecular target such as a protein.¹
• Splice-switching oligonucleotides (SSO), short, synthetic, antisense, modified nucleic acids that base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA-RNA base-pairing or protein-RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA.²
• Genome-editing drug moieties such as clustered regularly interspaced short palindromic repeat (CRISPR) combining a guide RNA (sgRNA or gRNA, a fusion of a CRISPR RNA and a trans-activating crRNA) CAS9 nuclease (CRISPR/CAS9), which function as a pair of molecular scissors, enabling genome editing by removing, adding or altering sections of the DNA sequence. Note that there are other (albeit more complex and less versatile) gene editing nucleases, such as zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), that also are being used for genome editing.³
• Locked nucleic acid (LNA) oligonucleotides that contain one or more nucleotide building blocks in which an extra methylene bridge fixes the ribose moiety either in the C3′-endo (beta-D-LNA) or C2′-endo (alpha-L-LNA) conformation.⁴
• Transgene, an exogenous gene introduced into the genome of another organism’s cistron, a segment of DNA that is involved in producing a polypeptide chain or otherwise modifying the activity of a cell (e.g., modify gene expression) to generate a desired effect.¹
• Chimeric antigen receptor T-cell (CAR-T), treatment in which a patient’s (autologous) or donor’s (allogeneic) T cells are collected, enriched for cells with specific cell characteristics, and/or genetically modified ex vivo to identify and kill cancer cells. Large numbers of the CAR-T cells are expanded in the laboratory and given to the patient by infusion.⁵
• Hematopoietic stem cells, immature cells that can develop into all types of blood cells, including white blood cells, red blood cells and platelets. Hematopoietic stem cells are found in the peripheral blood and the bone marrow.⁶
• Non-viral vectors, DNA, plasmids or mRNA that can be delivered to target cells as naked nucleic acid material or compounded with liposomes, gelatin and pegylated lipid nanoparticles (LNP) to provide better extracellular stability and improved cell penetration/delivery.⁷
• Viral vectors (VVs) viruses, such as adeno-associated viruses (AAV) and lentiviruses (LV), that transmit genetic material, DNA and RNA, into target cells, but also include other modified viruses such as herpes and vaccinia viruses, that have been modified to specifically infect cancer cells and either directly or indirectly (through induction of a patient’s immune response or by making the cells susceptible to a chemotherapeutic agent) kill the infected cells.⁸
• Replication competence assays (RCA or RCL when applied to LV vectors) required to ensure that the viral-vectored transgene therapy does not have the ability to recombine with other genetic material.
• Sanger and deep or next-generation (NG) sequencing used to demonstrate transgene identity, fidelity and integrity to the parent nucleic acid molecule. NG sequencing also is applied to identifying gene mutations within a target disease population.
• Quantitative and digital polymerase chain reaction (qPCR or dPCR, the later often described as ddPCR to identify the application as using a droplet reaction-based technology) used to confirm identity but more often applied to quantitate the number of molecules or genomes contained within a volume of drug product. These also can be used to evaluate the impact of gene therapies on targeted cellular expression in patients.

Having a common language and understanding of the key terms within the GCT space helps us better streamline our operations and serve our clients. Our bioanalytical lab has supported cell and gene therapies for more than 20 years with experts in ligand binding assays, activity assays, cell-based assays, chromatography, mass spectrometry, PCR, ELISPOT, next-generation sequencing, sanger sequencing, genomic arrays and flow cytometry. Our GMP labs also support extractable/leachable studies for products with unique features and materials associated with production and patient applications associated with CGT products. Our dedicated teams of scientists support CGT programs in our bioanalytical, GMP and central labs, and we are we continually expanding our capacity and capabilities to meet the needs of our growing client base and their CGT products.