Key implications for nonclinical development: FDA guidance on human gene therapy products incorporating human genome editing

This blog is part of The Regulatory Navigator series, where we explore the evolving regulatory landscape with actionable insight from Parexel's experts, sharing their experience to maximize success for clinical development and patient access.

The potential of human gene editing (GE) to change the lives of those with genetic diseases cannot be understated. With substantial growth in the development of therapies utilizing advances in GE technologies, in January 2024 the FDA released a draft guidance on human gene therapy products: Human Gene Therapy Products Incorporating Human Genome Editing.¹ The guidance describes the product information and nonclinical studies needed to support a successful IND submission for an investigational GE product.   

In this article, we discuss key takeaways from the guidance about FDA’s expectations for nonclinical assessment and identify important considerations for future GE product development. 

Current GE product landscape 

The current human GE product landscape is advancing rapidly and dominated by CRISPR/Cas9-based approaches. Ex vivo applications of CRISPR technology include, but are not limited to, the editing of T cells and NK cells for oncology and autoimmune indications as well as hematopoietic stem cells (HSCs) for genetic red blood cell disorders. In December 2023, the FDA approved the first CRISPR-based therapy in the US.² CasgevyTM (exagamglogene autotemcelis) is an ex vivo Cas9-modified HSC therapy for the treatment of Sickle Cell Anemia and B-thalassemia.  

The landmark approval of CasgevyTM has established a regulatory framework for the development and approval of future GE therapies. Utilizing this framework, other companies are rapidly advancing similar ex vivo CRISPR-edited HSC products for the same hemoglobinopathies. An example in vivo application of CRISPR/Cas9 is a program targeting transthyretin amyloidosis which recently initiated phase III testing. This program uses Cas9 to knock out the TTR gene to reduce misfolded circulating TTR protein and, consequently, the cardiac and neurological hallmarks of the disease. 

Second-generation variations of CRISPR technology involve engineering Cas9 to include a base-editing enzyme (e.g., deaminase) or a reverse transcriptase.³ These approaches, known as base editing and prime editing, respectively, combine the DNA-targeting ability of CRISPR with the ability to chemically modify a target DNA base or correct longer pathogenic sequences. Base-editing could be useful for treating conditions caused by common point mutations including Alpha-1 Antitrypsin Deficiency and Glycogen Storage Disease 1a, for example. In contrast, prime editing may prove useful for addressing mutation hotspots, repeat expansions, or eventually inserting whole genes, which has the potential to treat diseases including Friedreich’s Ataxia and Myotonic Dystrophy, among others.  

Additional human GE strategies in development include zinc finger nucleases (ZFNs), transcription activator-like effectors (TALENS), and homing endonucleases. For example, a homing endonuclease is in early development for in vivo GE applications targeting hepatitis B, among other indications.  

Overall, the multitude of GE programs in development will require thorough, complex, and product-specific regulatory considerations to support their advancement through preclinical and clinical testing. 

Key guidance takeaways for nonclinical development of GE products  

The overall goal for the nonclinical portion of this guidance is generally the same as that described in FDA’s guidance on preclinical evaluation of gene therapy products,⁴ which is to generate nonclinical data that supports the rationale and safety of the product in the intended clinical population.  

Important points from the January 2024 draft guidance on human GE products are: 

1. The GE product used in the nonclinical assessments should be as close as possible to the intended clinical product. In line with previous guidance, the nonclinical program should demonstrate the feasibility and safety of the intended clinical route and a pharmacologically active and safe dose level range. It should also establish the level of editing needed to achieve the intended therapeutic effect, including the minimum number of units (cells, etc.) that should be administered to achieve the desired effect. 
2. With proof-of-concept (POC) study expectations, the GE product should be tested in a system that demonstrates a biological response in the target patient population. As with other biotechnology-derived therapies, the agency recognizes that there are differences between human and animal model species. Thus, surrogates should be used when appropriate, provided a rationale for how the surrogate product supports the activity and safety of the GE product is included. Further, the POC studies should answer the following questions: Is editing at the desired locus achieved? Do the studies demonstrate the intended functional outcome?  
3. Biodistribution data bridges the gap between POC and safety data, and can be generated either stand-alone or as a part of a well-designed POC or safety study. As recommended by the FDA in previous guidance⁴, once the type, genomic location, and frequency of alterations caused by the GE product are identified, the biodistribution study should help to assess their impact on physiology. For example, how does the GE therapy impact cellular function, survival, proliferation, and differentiation capacity?  
4. Evaluation of on- and off-target editing events is of particular importance for nonclinical safety assessments. The emphasis on unintended on-target editing events is new and significant in this guidance and has most likely been added to recognize that safety issues may arise from unintended on-target editing events. For example, CRISPR-Cas9 has been shown to induce structural variants at on-target sites that can result in heritable mutations.⁵ Thus, it is important to characterize the nature and frequency of these on-target events to inform product safety and clinical monitoring. The FDA recommends that the potential for on-target editing be examined in multiple ways. In silico prediction tools such as BLAST are a suitable starting point, followed by appropriate biochemical and cellular assays. Further, both genome-wide and targeted approaches are recommended concomitantly. The nonclinical toxicology program should characterize toxicities, both local and systemic, as well as the timing of onset and resolution. The data is also expected to determine if there is a dose effect on the toxicology findings. The agency does not default to a certain species or to more than one species for toxicology evaluation but requires a scientifically comprehensive justification for the species selected. The agency will also expect a justification if no appropriate animal species exists. 

Altogether, a successful nonclinical program for a GE product should inform patient risk and provide screening and mitigation strategies for the proposed clinical study. The considerations above represent only a summary of the recommendations within FDA’s final guidance. We recommend reviewing the guidance in full to understand the implications for your human GE product development program.  

Future considerations for nonclinical evaluation of GE products 

In our interpretation of the recent guidance, the primary concern of health authorities evaluating GE products will be minimizing undesired, off-target editing within the genome of somatic tissues as well as avoiding on- and off-target mutations in the germline. The emphasis on minimizing unintended mutations highlights the importance of a thoughtfully executed preclinical program and product specificity toward both the genomic and tissue target. Even prior to the issuance of the finalized FDA GE guidance, we advised our clients who are developing in vivo GE products to focus their nonclinical program on evaluating candidate products that ultimately limit the in vivo duration of expression of the GE elements, when they have several options for delivery vehicle and route of administration. This principle is reinforced by the FDA guidance, which emphasizes the importance of limiting the period of functional activity for the GE component to minimize potential for both off-target and unintended on-target editing events. 

To address the safety concerns related to off-target editing highlighted in the recent guidance, major efforts are underway to develop next-generation Cas9 variants or orthologs with improved fidelity and genomic target specificity in addition to engineering tissue-specific delivery methods. Importantly, next-generation technologies may enable CRISPR-like therapeutic effects without the concerns of permanent genome editing. One such promising approach involves combining the DNA-targeting function of CRISPR-Cas systems with transcription and/or epigenetic factors to activate or repress gene expression at a target locus in what is termed “epigenome editing”.⁶ Furthermore, the discovery that certain Cas9 orthologs target single-stranded RNA has led to the development of RNA base editing strategies.⁷ The advent of epigenome and RNA editing offers potentially safer alternatives to GE, though these methods will still need to demonstrate target specificity and avoid off-target effects in the genome and/or RNA transcriptome. 

The future of GE technologies holds great promise for the treatment of human disease but is fraught with developmental and regulatory challenges. Considering that the regulatory guidance on GE will continue to evolve as more GE products advance through nonclinical and clinical testing, close consultation with regulatory subject matter experts is critical for successful program execution.  

Parexel’s regulatory consultants, who include former senior regulators and industry experts in cell and gene therapy products, are always available for a conversation to discuss your product development plans and support you in interpreting and actioning the latest guidance. Please get in touch.  

References 

1. FDA Guidance for Industry, Human Gene Therapy Products Incorporating Human Genome Editing (2024).  https://www.fda.gov/regulatory-information/search-fda-guidance-documents/human-gene-therapy-products-incorporating-human-genome-editing.   
2. FDA News Press Release, FDA Approves First Gene Therapies to Treat Patients with Sickle Cell Disease (2023).  https://www.fda.gov/news-events/press-announcements/fda-approves-first-gene-therapies-treat-patients-sickle-cell-disease. 
3. Pacesa M, Pelea O, Jinek M. Past, present, and future of CRISPR genome editing technologies. Cell. 2024 Feb 29;187(5):1076-1100. doi: 10.1016/j.cell.2024.01.042. PMID: 38428389. 
4. FDA Guidance for Industry, Preclinical Assessment of Investigational Cellular and Gene Therapy Products (2013). https://www.fda.gov/media/87564/download  
5. Höijer, I., Emmanouilidou, A., Östlund, R. et al. CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations. Nat Commun 13, 627 (2022). https://doi.org/10.1038/s41467-022-28244-5   
6. Hilton, I., D'Ippolito, A., Vockley, C. et al. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol 33, 510–517 (2015). https://doi.org/10.1038/nbt.3199  
7. Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, Cox DB, Shmakov S, Makarova KS, Semenova E, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV, Zhang F. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science. 2016 Aug 5;353(6299):aaf5573. doi: 10.1126/science.aaf5573. Epub 2016 Jun 2. PMID: 27256883; PMCID: PMC5127784. 

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