Gene Therapy.

Gene therapy is a cutting-edge medical treatment that aims to treat or cure a disease by transferring, altering, or removing genetic material in affected cells in the human body. The concept of gene therapy arose during the 1960s and early 1970s and was a direct consequence of the discovery of efficient DNA-mediated genetic transformation of cells and expression of foreign DNA having a desired function, that was made in the preceding years (Friedmann, 1992).

But it was not until the 1980s when gene therapy entered a new era following the discovery of retroviruses which proved a much more efficient tool for gene transfer. Richard Mulligan of the Massachusetts Institute of Technology developed the first viable retroviral vector for gene therapy. On January 19, 1989, the director of the National Institutes of Health (NIH), Dr. James A. Wyngaarden, approved the first clinical protocol to insert a foreign gene into the immune cells of persons with cancer (Roberts, 1989). On September 14, 1990, W. French Anderson and his colleagues at the NIH performed the first approved gene therapy procedure on a four-year-old girl, Ashanti DeSilva, born with severe combined immunodeficiency (SCID) (Anderson, 1990). Her treatment lasted twelve days, her blood cells were extracted, a new working copy of the ADA gene was introduced with an infective retroviral vector and cells were reinfused into her; overall, the procedure was similar to a bone marrow transplant. The goal was to replenish Ashanti’s blood cells with ones that could produce ADA. Ashanti improved so much she no longer needed to be kept in isolation, she is still alive today.

It took until the millennium before the first regulatory guidance emerged in the EU and the US. To date, guidance for quality, non-clinical and clinical development of gene therapy products are available as well as monographs in the European Pharmacopoeia and United States Pharmacopeia. According to the FDA, Human gene therapy “seeks to modify or manipulate the expression of a gene or to alter the biological properties of living cells for therapeutic use. Gene therapy is a technique that modifies a person’s genes to treat or cure disease.” The EMA defines gene therapies as advanced therapy medicinal products (ATMPs). “Gene therapy medicines contain genes that lead to 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, or long-term diseases. A recombinant gene is a stretch of DNA that is created in the laboratory, bringing together DNA from different sources.”

Over the past decades gene therapy has evolved in significant ways and new, more efficacious and safer vectors have been developed. Currently, vector based gene therapy products are being studied to treat diseases including cancer, genetic diseases, and infectious diseases, and as of February 2023, there are 13 approved gene therapy products in the US. According to the most recent EMA annual report, dated November 2022, the EU had 16. In February 2023 the European Commission granted Hemgenix market authorisation for the treatment of Haemophilia B, bringing the total to, at least 17.

Gene Editing.

Now we are facing a new era where the DNA can be modified with a precision never seen before thanks to directed gene editing. Since the discovery and development of zinc finger nucleases in the early 2000s, gene editing technologies have been increasingly studied.  Though pioneering, this initial approach was impeded by the difficulties of building zinc finger arrays that could target all sections of the genome. In 2009, transcription activator-like effector nucleases (TALENs) took their place, allowing the production of unique TALENs capable of altering practically any gene. TALEN proteins are simpler to develop and provide more precision in editing technologies, with less off-target impacts. Later, the CRISPR-Cas9 system in bacteria was identified and exploited. CRISPR allows guide RNAs to direct the Cas-9 protein to practically any location in the DNA. In later versions of the system, DNA may be cut, sections deleted, and new sections introduced to modify the proteins generated. The Nobel Prize was awarded to CRISPR in 2020 in recognition of its potential widespread application for treatments. It is the CRISPR system that has been employed to target diseases known to occur from a single nucleotide mutation.

The adaptability of CRISPR-Cas to target a particular sequence in the genome is an advantage of the technology over ZFN and TALEN for gene editing. Unlike TALEN and ZFN, where it is necessary to engineer a new targeted nuclease for each target sequence, a process that can take weeks to months due to the complexity of recombinant protein expression and purification, the target specificity of Cas9 and Cas12a is entirely determined by the associated guide RNA, which can be readily synthesised.

CRISPR Mechanism.

CRISPR/Cas9 is a two-component system composed of Cas9, an RNA-guided endonuclease capable of cleaving double-stranded DNA, and a 20-nucleotide trans-activating RNA (tracrRNA), which is directed to a target sequence by a 20-nucleotide complementary sequence in the CRISPR RNA (crRNA). The crRNA and tracrRNA can be joined by a tetraloop to form a single guide RNA (sgRNA) which directs the Cas9 to a target site for DNA cleavage.

When a sgRNA:Cas9 ribonucleoprotein binds its target sequence in the presence of a flanking protospacer adjacent motif (PAM) sequence, the Cas9 protein will introduce a double strand break (DSB). If a PAM sequence is lacking, the Cas9 will not cut. Cas9 proteins from different bacteria recognise different PAM sequences.

Applications.

The CRISPR/Cas technology allows for a targeted modification of the genomic material of a cell. From a product development standpoint there are two main uses of CRISPR/Cas: as a manufacturing tool or as a medicinal product itself. 

As a manufacturing tool, CRISPR gene editing has the potential to greatly improve antibody manufacturing processes, to engineer cells to grow at lower temperatures or in less expensive media to decrease production costs, and to modify biological pathways within cells to ensure adequate expression, post-translational modification, and folding of the resulting products. CRISPR may be employed on a widespread basis to modify the genomes of host cells to generate lines suitable for large-scale production. A number of firms have obtained a licence from ERS Genomics in order to utilise CRISPR for this specific purpose. The insertion of an antibody cassette into a single location or several sites within the genome of a cell may also be precisely regulated using CRISPR. This method reduces the potential for epigenetic silencing effects and promotes consistent gene expression at high levels. Additionally, it shortens the time required to clonally separate high-producing cells, which speeds up the development of new antibody-producing lines. In the process of drug development, one of the most common applications of CRISPR-Cas gene editing is knock-out screening utilised in identify genes involved in drug resistance. In this process, scientists expose a large number of cells to a pool of CRISPR-Cas systems containing target-specific guide RNAs. Thus, they may generate and isolate cells with a specific gene deletion. CRISPR-Cas-modified cells that can become responsive to drugs are an effective method for identifying the genes responsible for drug resistance. Utilising medicines that target these genes or the proteins these cells produce is another option for overcoming pharmaceutical resistance.

As a therapeutic product, the CRISPR/Cas9 system can be delivered in the form of a DNA plasmid encoding both the Cas9 protein and sgRNA, or it may be delivered in the form of Cas9 mRNA or native Cas9 protein, in which both cases the sgRNA needs to be co-delivered as well. Viral vectors, such as lentiviral vectors, adenoviral vectors, and adeno-associated virus (AAV) vectors have been used most often for the delivery of Cas9/sgRNA-encoded plasmids for CRISPR therapeutics, showing excellent gene transfection efficiency. However, viral expression can result in immunogenic responses, long-term expression failure, among other drawbacks. Moreover, a large fraction of the human population has pre-existing immunity to AAV, making them ineligible for AAV-based therapies. A panel of nonviral vectors is being developed to address the limitations of viral-based vectors and improve genome editing for both in vitro and in vivo applications. Cationic liposomes (CLs) are among the most promising vectors for delivering nucleic acids to cells.

The controls that have to be put in place depend on the use of the technology. When it is used as a manufacturing tool it has to be mainly controlled as part of the Quality/CMC program, when it is used as a medicinal product it has to also be controlled as part of the nonclinical program. But regardless of its use, there is a common risk that has to be assessed: the risk of off- target effects i.e., the risk of CRISPR/Cas altering sequences other than the target sequence.

CRISPR Challenges.

The first CRISPR-based therapeutic study in the United States (NCT03399448) combined CAR-T and PD-1 immunotherapy techniques, editing three genes in total with CRISPR. The objective of this study was to assess the safety and feasibility of multiplex CRISPR-Cas9 gene editing of T cells from patients with advanced, refractory cancer and determine whether the medication had tolerable side effects. In vivo tracking and persistence of the engineered T cells was monitored to determine if the cells could persist after CRISPR-Cas9 modifications. The editing efficiency was consistent in all products and varied as a function of the single guide RNA (sgRNA). Off-target effects were uncommon. Unintended edits at the target site, on the other hand, were common, with 70% of cells revealing at least one mutation at or near the target site during the T cell production process. The mutations induced by CRISPR-Cas9 were highly specific for the targeted loci; however, rare off-target edits were observed. Single-cell RNA sequencing of the infused CRISPR-engineered T cells revealed that ~30% of cells had no detectable mutations, whereas ~40% had a single mutation and ~20 and ~10% of the engineered T cells were double mutated and triple mutated, respectively, at the target sequences. The overall fraction of cells with mutations in patients reduced after infusion and throughout the trial. Although the investigation found a large percentage of significant genomic rearrangements, the proportion of cells with these mutations reduced over time. 

Cells with unintended mutations are thought to have perished or been outcompeted by other cells. There were no clinical toxicities associated with the engineered T cells. Chromosomal translocations were observed in vitro during cell manufacturing, and these decreased over time after infusion into patients (Stadtmauer et al., 2020).

Regulatory approach to off-target effects.

No guidelines have currently been set for methods to assess off target effects of these gene editing products or the level of off target data that must be generated to prove the safety and efficacy of a gene editing product.  This has been the topic of both EMA and FDA workshops to foster discussions on understanding different techniques to identify off target edits, as well as on target efficiency. In 2018 an EMA workshop included presentations from Editas medicines and Sangamo Therapeutics. The roundtable discussion concluded that in silico methods do not provide a reliable risk assessment alone and that experimental data targeting identified sites in in vitro models can provide useful information (EMA, 2018). Therefore, a combination of methods should be used, with regulators finding a need to compare and interpret evidence from animal and human data. 

The first part of the necessary data package will be the insilico design of the guide RNAs. The choice of guide RNA and their predicted off target effects are essential in minimising the chance of off target effects in vitro and in vivo. Any potential sites identified by in silico predictive tools for these final guide RNAs should be screened in vitro to ensure guide RNA stringency. 

Several in vitro techniques have been developed to ensure editing stringency. These methods include but are not limited to Guide seq, Digenome-seq, Site-seq and Circle-seq. The choice of technique must be justified to regulators, including its limitations and steps undertaken to combat these.  

A technique recently presented at an FDA CBER workshop on ‘Facilitating End-to-End Development of Individual Therapeutics’ was the use of a two-step method to identify and verify off target effects (CBER FDA, 2020). The first, a discovery phase in a surrogacy setting, using the ONE-Seq method, which uses purified genomic DNA and purified nuclease to identify all potential off target sites, before the second step of verifying if these exist in a therapeutic setting. 

This approach was undertaken by CRISPR Therapeutics for their CTX001 pre-clinical trial data package, which used extensive deep next generation sequencing at thousands of potential homologous sites, or putative sites identified by Guide-Seq, as well as a lack of safety signals in extensive biodistribution GLP toxicology studies. This ex vivo genome edited product is in Phase 1/2 trials. Similarly, EDIT 101 also used a discovery and verification phase to identify off target sites through in silico prediction, Guide-Seq and Digenome-Seq (Maeder et al., 2019). These were verified in in vitro assays using relevant cell lines as well as in human retinal explant tissue from two donors. 

Despite the challenges, gene editing with CRSPR/Cas systems as therapeutic products is becoming a reality. According to https://clinicaltrials.gov there are, at least 36, active, enrolling, and recruiting CRISPR trials ongoing, and, at least, 8 completed trials worldwide. Regulators are learning as developers advance in this exciting though challenging technology. At Scendea we have a team of leading experts in the CMC, nonclinical, and clinical regulatory aspects of gene therapy medicinal products, in regular contact with regulators and agencies, and who are ready to help support you at any stage of development.

REFERENCES

Anderson, W.F. (1990). September 14, 1990: The Beginning. Human Gene Therapy 1, 371–372.

CBER FDA (2020). WORKSHOP: Facilitating End-to-End Development of Individualized Therapeutics.

EMA (2018). Report of the EMA expert meeting on genome editing technologies used in medicinal product development.

Friedmann, T. (1992). A brief history of gene therapy. Nature Genetics 2, 93–98.

Maeder, M.L., Stefanidakis, M., Wilson, C.J., Baral, R., Barrera, L.A., et al. (2019). Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nature Medicine 25, 229–233.

Roberts, L. (1989). Human Gene Transfer Test Approved. Science 243, 473–473.

Stadtmauer, E.A., Fraietta, J.A., Davis, M.M., Cohen, A.D., Weber, K.L., et al. (2020). CRISPR-engineered T cells in patients with refractory cancer. Science 367, eaba7365.

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