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DDReg Pharma

The Advancements of Gene Editing Technology for Safe and Effective Gene Therapy Products

The field of gene therapy has witnessed significant advancements with the development of genome editing technologies. These technologies allow scientists to break, modify, and edit specific genes in a DNA sequence-specific manner, opening up new possibilities for gene therapy. While traditional gene therapy involves the addition of new genes to human cells or tissue, genome editing technologies have the potential to knock out or modify specific disease-causing genes, making them a promising tool for gene therapy.

Genome editing technologies rely on the introduction of double-strand breaks (DSBs) into specific DNA sites and the subsequent repair mechanisms of cells. Two main repair mechanisms involved in DSB repair are non-homologous end joining (NHEJ) and homology-directed repair (HDR) through homologous recombination (HR). NHEJ is a rapid response mechanism that occurs throughout the cell cycle and can cause the loss of target genes. On the other hand, HR occurs mainly in the S and G2 phases of the cell cycle and repairs DNA through recombination with a homologous sequence. HDR can be induced by introducing a DNA template carrying the desired sequence, which corrects the abnormal gene associated with the disease.

Types of genome editing technologies

There are several types of genome editing technologies, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems. ZFNs and TALENs are artificial restriction enzymes that cause DSBs specifically at DNA sequences. However, their manufacturing requires sophisticated technology, time, and a significant amount of money. On the other hand, the CRISPR/Cas system uses a single-guide RNA (sgRNA) to recognize the DNA sequence of a target gene. The design of CRISPR/Cas is easy to use and cost-effective, leading to its rapid development as a versatile genetic engineering technique.

Clinical trials using these genome editing technologies for gene therapies targeting infections, cancers, and single-gene diseases have been conducted overseas, and similar trials may soon begin in Japan. As genome editing technologies advance, it is crucial to clarify the concept of the quality and safety of gene therapy products utilizing genome editing.

Gene therapy products using genome editing techniques can be categorized into two main types: in vivo genome editing products and ex vivo genome editing products. In vivo genome editing products involve the direct administration of a gene therapy product using a genome editing technique in the body, while ex vivo genome editing products are human cell-based products manufactured by ex vivo genome editing using a genome editing tool.

Cancer Risk of Gene-Edited Cells

Genome editing technologies carry the inherent risk of off-target effects, which refers to unintended editing of genes that have similar DNA sequences to the target gene. Off-target effects can potentially result in the tumorigenicity (cancer) of cells. These effects may directly activate oncogenes or inactivate tumor-suppressor genes. Additionally, genome editing has the potential to cause permanent alterations in the genome, leading to genome instability and the risk of chromosomal breakage.

Risk of Unintended Gene Modification in Germline Cells

In vivo genome editing, which involves the direct administration of a genome editing gene therapy product in patients, may unintentionally result in the genome editing of off-target cells or modification of off-target genes. This is particularly concerning when genome editing is performed in pediatric patients or patients of reproductive age, as it may affect germline cells. The potential genetic effects in subsequent generations should be fully understood and justified. New technologies that allow genetic engineering without genomic cleavage have been developed to avoid the risk of chromosomal mutation. However, the effects of these technologies on the next generation should be carefully evaluated.

Cautions Regarding Genome Editing Tools and Gene-Modified Cells

When using genome editing tools, such as ZFNs, TALENs, or CRISPR/Cas, several factors need to be considered to ensure safety and efficacy. For viral or plasmid vectors used in genome editing, the quality control and characterization processes should be evaluated similarly to current gene therapy products. The potential risk of tumorigenicity associated with the insertion of viral promoter sequences adjacent to cancer-related genes should be addressed. The persistence of genome editing enzymes and their effects on off-target cells should also be evaluated.

For mRNA-based genome editing products, the manufacturing method and quality control need to be clarified, as mRNA products have not yet been approved for marketing. Chemical modifications of mRNA, such as methylated Cap, should undergo safety evaluations. Similarly, genome editing protein products should be evaluated as gene therapy products, considering their potential off-target effects and adverse events. The quality attributes of artificial nuclease proteins and sgRNA should be thoroughly evaluated.

Human cell-based products manufactured through ex vivo genome editing should undergo quality control and characterization processes similar to current gene-transfected cells. The safety of administering genome-edited cell-based products should be assessed through nonclinical safety evaluations.

Evaluation of Safety and Risks

Safety evaluation of gene therapy products using genome editing techniques should focus on specific issues, such as off-target effects, genome deletions/insertions, DNA-repair gene mutations, and the risk of cancerization among target cells.

Off-target effects can be characterized through experimental methods that explore candidate off-target sites throughout the entire human genome. The frequency and effects of off-target effects should be analyzed using whole-genome sequencing and amplicon sequencing. The risk of genome deletions/insertions and chromosomal translocations/inversions should be assessed through G-band analysis, mFISH, and CGH. The occurrence of gene mutations related to DNA-repair genes should be investigated, with a particular focus on p53 mutations. Differentiated cells are likely to have a lower risk of cancerization compared to iPS/ES cells and hematopoietic cells.

Immunogenicity of genome editing enzymes derived from bacteria should be considered, as they may be recognized as heterologous antigens in human cells. The potential immunotoxicity and immune response to these enzymes should be taken into account during clinical trials.

In Vivo Genome Editing

In vivo genome editing involves the direct administration of a genome editing product in the body. Safety evaluations should focus on the modified target genes, targeting and modification efficiency of genome editing enzymes, and other relevant factors. Animal studies may not provide sufficient information about off-target effects, so in vitro analyses using human cells should be conducted. Long-term follow-up plans, including periodic examinations, should be established to identify any adverse events related to genome editing.

Important Issues in Clinical Trials and Long-Term Follow-Up

Clinical trials of genome editing technologies should consider the potential risks associated with each technology, the type of target cells, and the targeted gene. Long-term follow-up is crucial for gene therapies utilizing genome editing, especially for hematopoietic stem cell-based therapies, which have a higher risk of adverse events. Germline gene modification should be carefully monitored, and measures should be taken to prevent modifications from affecting subsequent generations.


The advancements in gene editing technology have opened up new possibilities for safe and effective gene therapy products. However, as the field continues to evolve, ongoing research and evaluation of the safety and efficacy of genome editing techniques are essential. By carefully considering the potential risks and implementing long-term follow-up plans, gene therapy products utilizing genome editing can be developed and reviewed with confidence. These advancements have the potential to revolutionize the field of gene therapy and provide new treatment options for various diseases.

As part of its pharmaceutical product portfolio, DDReg provides regulatory solutions for biotechnology products along the entire product lifecycle. With experience of over 120 regulatory agencies and subject matter expertise, DDReg is the go-to-partner for pharmaceutical regulatory services. Read more from the experts: Ensuring Viral Safety of Biotechnology Products

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