CRISPR/Cas9 gene-editing technology is facilitating the development of novel cell and gene therapies. Aldevron is helping speed up the process by making standard nucleases available for the production of research, clinical trial and commercial products.
Gene-Editing Technologies
Gene-editing technology has advanced dramatically in the last two decades. The ability to directly manipulate genes across cell types was first realized via the engineering of chimeric nucleases comprising sequence-specific DNA-binding domains fused to non-specific DNA cleavage modules. These chimeric nucleases — zinc-finger nucleases (ZFNs) and transcription-activator like effector nucleases (TALENs) — generate permanent mutations via the introduction of sequence-targeted double-stranded breaks (DSBs) that activate DNA repair pathways. These methods are costly and time-consuming, however, and not generally practical for large-scale manufacturing.
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats with CRISPR-associated protein 9) technology presents a faster, cheaper, more accurate and more efficient gene-editing method that affords targeting efficiencies similar to those obtained with TALENs and ZFNs. In addition, it allows for site-specific genomic targeting in virtually any organism.
The technology takes advantage of a naturally occurring gene-editing system that bacteria use to “remember,” target and disable invading viruses. The CRISPR/Cas9 system comprises the Cas nuclease for cutting DNA and single-guide RNA (sgRNA) or both CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA) for binding to the target DNA sequence and to Cas9. The cleavage of double-stranded DNA activates the DSB repair machinery in a cell, which can involve the cellular non-homologous end-joining (NHEJ) pathway, which leads to insertions and/or deletions or the homology-directed repair (HDR) pathway, which makes precise replacement mutations.
This simple system is being widely employed in the development of novel medicines and many other industrial applications. Roughly three years ago, the National Institutes of Health in the United States awarded $300 million in funding for research projects using CRISPR/Cas9 gene-editing tools. By 2018, over $1 billion in funding was awarded. This investment has led to the publication of more than 3,000 peer-reviewed journal articles exploring the CRISPR/Cas9 system and its applications.
In the pharmaceutical sector, CRISPR/Cas9 is largely being used to develop treatments for genetic diseases — there are over 6,000 monogenetic diseases caused by the mutation of a single base pair in the genome. The gene-editing technology is also being leveraged for the development of treatments for a host of other maladies, such as cancer, Alzheimer’s disease and numerous other disorders.
Recent Innovations
Gene editing is a nascent field, and much effort continues to be focused on improving the performance of the CRISPR/Cas9 system. While most Cas9 systems use the nuclease from Streptococcus pyogenes (SpCas9), Cas9 nucleases from different hosts have also been explored. For example, the Cas9 enzyme from Staphylococcus aureus (SaCas9) is smaller than SpCas9 while providing similar functionality, making it easier to package into viral vectors.
Other advances in CRISPR/Cas9 technology have resulted from the engineering of mutations of wild-type Cas9. In the SpyFi™ Cas9 nuclease developed by Integrated DNA Technologies, Inc. (IDT), a single amino acid replacement results in a significant reduction of off-target effects, which is a key safety concern for many gene and cell therapies.
Modified versions of the CRISPR/Cas9 system with an added second enzyme also have been developed that serve as base editors. Rather than performing a double-stranded cut of the target DNA, the system modifies a DNA base pair itself, resulting in a cytosine change to a guanine or adenine change to a thymine.
Designed for GMP Production
As an established contract development and manufacturing organization, Aldevron has experience producing thousands of proteins, including hundreds of lots of different gene-editing enzymes, including CRISPR/Cas9 nucleases, such as SpCas9 variants, SaCas9, endonuclease-dead (dCas9) fusions and nickases. Custom nucleases are supported by robust, scalable manufacturing protocols, including the capability of internally transferring from research grade production to Aldevron’s recently built cGMP facility; our manufacturing scale ranges from 10 mg to multi-gram lots.
When developing new gene-editing tools for use in the production of cell, gene or other therapies, it is essential to consider the requirements for GMP-compliant manufacturing from the outset. Aldevron’s extensive experience in the gene-editing field has provided us with the expertise needed to engineer nucleases with desirable attributes.
For instance, at the research stage, it is often useful to attach affinity tags to enzymes to enable the use of affinity capture chromatography as a purification method. However, leaching of nickel and other divalent cations from the chromatography column into the Cas9 protein can occur. While trace quantities of such impurities will typically not be an issue for use in research applications, they are not acceptable in nucleases used in CRISPR gene editing for drug production.
Aldevron can help clients at the preclinical stage transition from a process suitable for laboratory scale to one that is applicable for GMP production of material for human trials and ultimately commercialization.
Further Simplification of Gene Editing
Part of our growth strategy is to advance the gene-editing field by providing integrated services and adding value for clients through various product offerings. Custom production of Cas9 nucleases can take an estimated 6–12 months from initial method development to GMP product delivery. In many cases, an additional six months of wait time may be added to that timeline, if manufacturing capacity is limited or a new assay needs to be validated.
The production of bulk gene-editing nucleases as standard, off-the-shelf products for research, clinical trial and commercial manufacturing was a solution Aldevron was able to deliver to clients. It has been shown, in fact, that using Cas9 in protein form provides many benefits, including reduced off-target effects, immediate intracellular activity and controlled dosage capabilities.
Currently, we offer four standard nuclease products: SpyFi™ Cas9 Nuclease, SpCas9 Nuclease, AsCas12a Nuclease (Cpf1) and SaCas9 Nuclease. When used in combination with an effective guide and specific target sites, SpyFi Cas9 Nuclease — licensed from IDT — delivers clinically relevant on-target editing efficiency with reduced off-target effects when compared with wild-type SpCas9 nuclease.
All of these in-stock products are offered as Research Grade, GMP-SourceTM and GMP formulations for immediate delivery. They are engineered to be used in a variety of applications, including electroporation, transfection and microinjection and are prepared at a concentration of 10 mg/mL. On a per-project basis, custom variations are also available, including custom concentrations, formulations and QC testing.
Accelerating Development
Most research-grade CRISPR-associated nucleases are not applicable for clinical applications due to their construct designs and quality grade. Aldevron’s Research Grade nucleases, however, have the same construct design as our GMP-SourceTM and GMP products. Notably, our Research Grade, GMP-SourceTM and cGMP Cas9 nucleases exhibit the same editing efficiency.
Our Research Grade material is produced rapidly but with high quality (although not sufficient for use in humans), enabling the advancement of preclinical research programs. GMP material is produced in a purpose-built suite designed specifically for high-quality manufacturing under conditions that are compliant with current Good Manufacturing Practices. The suite has its own air handling system with extensive environmental monitoring and oversight through the presence of a second operator serving as a verifier. Aldevron’s clients have determined that this material is of sufficient quality to be used to manufacture material for clinical applications.
GMP-Source™ nucleases provide a more rapid, cost-effective sourcing option for cell and gene therapy projects. They allow seamless transition from research to a cGMP product because they retain the key hallmarks of cGMP manufacturing but with reduced costs and development timelines. As a result, it is possible to save money and time.
By offering this interim quality grade, we are helping cell and gene therapy developers implement phase-appropriate GMP-compliant solutions. Indeed, Aldevron CRISPR-associated nucleases are currently being used in multiple clinical trials.