CRISPR RNPs and the Future of Cell and Gene Therapy

As clustered regularly interspaced short palindromic repeats (CRISPR) therapies progress to the clinic, the key CRISPR editing components are primarily being delivered by recombinant virus, RNA, or as ribonucleoprotein (RNP), which is the active complex formed by the guide RNA and Cas9 protein. RNPs have several attractive properties as a delivery format, particularly in the manufacturing of ex vivo cellular therapies, the most rapidly growing segment of the CRISPR therapeutics market.

To support this demand, Aldevron has developed a comprehensive process for manufacturing CRISPR RNPs for clinical applications, including assays to optimize complexing conditions, to analytically measure cutting activity, and to quantify the content of bound and unbound components. Stability of the RNPs has been confirmed for up to 18 months under proper storage conditions, and longer studies are currently underway. This work has enabled the company to offer a complement to its GMP Cas9 protein products and a much-needed path to custom GMP RNP material for developers of CRISPR-based therapies.

Genome Editing vs. Gene Therapy

Many diseases are caused by errors in the genetic code, which can include missing, extra, and dysfunctional genes. With gene therapy, missing or dysfunctional genes are supplemented with the corrected form of the gene, administered either directly into the patient or first into cells ex vivo that are then transferred to the patient.

The mutations that cause some genetic diseases cannot be remedied with gene therapy. Fortunately, the science of genome editing, developed in research labs over the last two decades, has advanced to the point where it is now rapidly progressing through the clinic in the form of numerous therapies. These genome-editing treatments directly modify the DNA of the cell to cure or alleviate diseases with a genetic basis. As with gene therapy, genome editing can take place ex vivo in cell culture or within the patient.

Technology Advances Spur Expansion of Genome Editing Market

The global market for genome editing/genome engineering is predicted to expand at a compound annual growth rate of 15–17%, reaching $11.2 billion in 2025, according to MarketsandMarkets,¹ or $9.2 billion by 2026, according to Acumen Research and Consulting.² 

These estimates include numerous genome-editing technologies (e.g., CRISPR, transcription activator-like effector nuclease (TALEN), zinc-finger nuclease (ZFN), and others) used for cell line editing, animal genome editing, plant genome editing, and the development of human therapeutics.

While research-use and animal genome engineering currently dominate the market, investment by pharma and biotech companies to develop new drugs against previously intractable diseases is a key growth driver.² Most are leveraging CRISPR because it is easy to use, provides high throughput, and is more affordable than alternative technologies. Reliable sources of GMP CRISPR components will be an important factor in realizing the growth of the therapeutic market.

The first U.S. human clinical trial for a CRISPR-based therapy was initiated in April 2019 and many more have been initiated since in the United States, Canada, Europe, and China. The candidates advancing furthest in the clinic to-date modify immune cells to treat cancer, correct a blindness-causing mutation in retinal cells, and restore normal function to the red blood cells of patients with hemoglobin-based diseases, like sickle cell disease and beta-thalassemia.

Three Main CRISPR Delivery Formats

In current approaches to clinical genome editing, CRISPR is used to inactivate (or “knock out”) genes by disrupting their instructions or to introduce new genetic sequences to the genome (known as “knock-in”). The CRISPR-associated (Cas) nuclease, exclusively Cas9 in current clinical trials, does the physical cutting of the genome, while the guide RNA (gRNA) directs Cas9 to the intended DNA target. Both of these components must be efficiently and simultaneously delivered to the DNA in the nucleus of patient cells in order for them to perform their function.

Currently, there are three formats primarily being used for the clinical delivery of Cas9 and gRNA: virus, RNA, and RNP (complexed gRNA and Cas9 protein).

By their very nature, many viruses are adept at entering mammalian cells. Researchers have taken advantage of this ability and turned viruses into therapeutic delivery devices by removing viral genes responsible for disease and replication and replacing them with desired genes, such as those that make Cas9 and gRNA. Researchers choose the type of modified virus to use in their therapy based on one or more desired attributes, such as targeting a specific type of tissue, avoiding the immune system, or permanently integrating the therapeutic genes carried by the virus into the host genome. Viruses chosen to deliver CRISPR-based therapies are selected for their ability to easily enter mammalian cells. While viruses offer some advantages, especially for in vivo delivery, they also have some distinct drawbacks. Manufacturing GMP-grade recombinant virus is extremely expensive, complex, and time-consuming. Standardizing viral dosage is a challenge from patient-to-patient and lot-to-lot, and, once the virus is inside the cell, the expression of Cas9 and gRNA is constant and uncontrolled until the viral genome has been degraded. The extended presence of the CRISPR components in a cell is strongly correlated with an increase in off-targeting, or the cutting of the wrong DNA sequences. Additionally, the genes delivered by the virus require transcription and translation, so there is a delay between the delivery of the reagent and the onset of editing activity. There is also the possibility of unintended integration of the viral genome into the patient’s genome, although this seems to happen at low frequencies. Finally, viruses are fairly limited in the amount of genetic information they can hold, restricting the options for a genome editing therapy.

Delivery of the CRISPR components as RNA (mRNA for Cas9 and synthesized RNA for the gRNA) has some advantages over viral delivery. RNA is titratable to a much greater degree than uncontrolled expression from a viral vector, since each RNA molecule represents a single Cas9 protein or gRNA molecule, allowing precise dosing. Fortuitously, many of the nanoparticle systems developed for in vivo siRNA delivery in the past have been easily modified to accommodate in vivo CRISPR RNA delivery, accelerating the progress in this area. There are concerns, however, regarding the immunogenicity of exogenously delivered synthetic RNA. RNA-based delivery also requires translation of the Cas9 mRNA into protein, a step that is not required for the gRNA; this decoupling may confound efforts at exact titration, given the rapid degradation of RNA in cells. Finally, while nanoparticle formulation has given CRISPR RNA formats the potential for in vivo delivery, problems of tissue specificity still remain to be overcome.

Given these issues, RNPs currently present the most attractive approach for delivering CRISPR components into cells. RNPs provide a DNA-free delivery system without the need for transcription or translation. Because they are composed of the Cas9 protein and gRNA, RNPs are immediately active, so there is no delay in editing. Though they cut immediately, the gRNA and Cas9 are rapidly cleared from cells (fast-on/fast-off kinetics), reducing the risk of off-target cutting activity. Furthermore, similar to classic small molecule drugs, each RNP represents a distinct unit of activity, making them highly titratable and allowing for precise dosage and controlled delivery. RNPs can also be delivered at much higher efficiencies to cells that are difficult to transduce with viruses.

For current CRISPR therapies, RNPs are being delivered into cells ex vivo, most commonly via electroporation. In the immuno-oncology space, CRISPR RNPs are being used to modify a range of genes, including those coding for native T-cell receptors, genes involved in tumor suppression, such as PD-1, or HLA genes, which trigger the graft-versus-host response common in tissue transplants. Removing the correct combination of HLA-related genes may open the door to universal off-the-shelf allogeneic cellular therapies derived from a single donor. CRISPR RNPs are being used in many other disease areas as well, creating a significant demand for clinical RNP material and supporting characterization assays.

A Growing Number of Options

A looming challenge to manufacturing clinical CRISPR RNPs lies in the vast array of CRISPR/Cas proteins currently under development. In addition to the many Streptococcus pyogenes Cas9 variants available on the market today with slightly different nuclear location sequencers (NLSs) — for instance, there are up to 50 NLS variants of SpCas9 — substantial development has gone into improving the genome-editing profile of the SpCas9 enzyme, either through rational design or directed evolution experiments, resulting in a suite of enhanced SpCas9 nucleases. Continued investigation has also uncovered the existence of CRISPR enzymes with unique properties residing in other bacterial strains, and many of these orthogonal Cas proteins are being developed into genome-editing tools, creating even more options when choosing a CRISPR system. In addition to modifying the Cas9 nuclease itself, numerous Cas9 fusion proteins have also been demonstrated, including combinations of nuclease-deficient (“dead”) SpCas9 with activator, inhibitor, acetylation, methylation, deaminase, and reverse transcriptase domains, to name a few.

While some Cas9 fusions are easy to produce as a purified protein, others are more challenging. The linker for fusion proteins must be carefully selected; if it is too short, the Cas9 might not be able to physically bind to the genome, and if it is too long, it may not efficiently associate with the target DNA. Furthermore, many of the novel Cas variants and fusions have been characterized using plasmid-based expression, generating results and activity profiles that are not representative of the formats through which a CRISPR therapeutic would be administered. Modeling these Cas fusions as RNPs provides the truest assessment of their performance.

As these variations on the CRISPR system progress towards clinical development, the conditions for complexing and characterizing each Cas variant or fusion protein with its gRNA will require optimization specific to that system. For instance, factors, such as the ideal ratio of gRNA to protein, temperature gradient, complexing time, and other parameters, must be individually determined for each combination of a new Cas protein and its guide RNA. Importantly, for off-the-shelf therapies to become a reality, RNPs must be stable for extended periods of time. Real-time studies using both in vitro and in vivo assays must be performed to ensure that these materials remain effective during storage. Considering the growing number of Cas9 variants and fusions, the magnitude of development required to support these disparate systems can be daunting.

From Simple Mixing to Optimized Production 

Prior to Aldevron’s development work, most CRISPR RNPs used in clinical processes were generated by simply mixing the Cas9 protein and gRNA together and letting the mixture sit for a few minutes before applying it to cells. While this approach seemed to be sufficient for research models, there was little evidence that this process had been optimized for therapeutic applications, where maximizing effectiveness and efficiency is critical. It was in response to requests from our therapeutic partners that Aldevron began investigating the optimal conditions for combining the Cas9 protein and gRNA. As a result of these efforts, Aldevron has created a novel process to complex, QC, and characterize RNPs produced from GMP-grade Cas9 proteins and GMP-grade chemically synthesized single guide RNAs (sgRNAs) under GMP conditions.

In order to determine optimal complexing conditions, Aldevron first had to develop a platformable in vitro cutting assay to measure relative RNP activity. Using this assay, it is possible to rapidly assess a range of gRNA:Cas9 complexing ratios and define absolute cutting activity, the linear range of cutting, and the limit of detection for a specific RNP complex. The results of the in vitro cutting assay are then compared and correlated to the RNP’s activity at the endogenous DNA target for a specific cellular model. Using our proprietary in vitro cutting assay, Aldevron found that maximum cutting is typically achieved at a gRNA:Cas9 protein ratio of 1.5–2 molecules of gRNA per Cas9 protein molecule; these findings were corroborated in cellular assays for RNP cutting activity.

Anticipating Regulatory Requirements

Given the rapid advances in the development of CRISPR-based therapies, it is unsurprising that regulatory bodies have lagged behind in providing relevant guidance on the manufacturing, QC, and validation process for CRISPR systems used in clinical applications. In anticipation of these future regulatory structures, Aldevron is working closely with our therapeutic partners to characterize our GMP-produced CRISPR RNPs to the fullest extent possible.

One of the primary considerations for formulating a new drug is to define the quantity and function of each component in the composition. In order to assess the contents of the CRISPR RNP composition, Aldevron developed novel assays to quantify the levels of free (unbound) Cas9 and gRNA, relative to fully formed RNP. To detect free Cas9, differential scanning fluorimetry and size-exclusion chromatography were initially assessed and found to be ineffective for either the separation or quantitation of the individual components. However, analytical cation exchange chromatography (aCEX) was found to be capable of distinguishing and quantitating free Cas9 in the RNP mixture. Notably, it is essential to use high-purity (HPLC or PAGE purified) sgRNA for this assay, as the RNP segregates from free Cas9 based on the charge of the bound gRNA; lower-purity gRNAs carrying a spectrum of truncation products can erase this separation, confounding the assay. Native polyacrylamide gel electrophoresis (PAGE), meanwhile, was found to be a reliable method for separating and quantifying unbound sgRNA. 

Next, we conducted long-term stability studies to determine the shelf-life and optimal storage conditions for RNPs produced using our complexing process. In order to assess ideal storage conditions, RNPs were maintained at –80, –20, 4, and 30 °C for up to six months and at –80 °C for up to 36 months. At each time point, the RNPs were assayed for cutting activity, and our panel of QC assays was used to determine the quantity and stability of the individual components. Not surprisingly, significant degradation of the RNP with a concomitant decrease in cutting was observed after six months of storage at 30 °C, but no significant degradation or reduction in performance was observed after six months at the other three temperatures.

After 18 months, robust cutting activity was maintained for the material stored at –80 °C, similar to the activity observed on day one, with no signs of RNP, free Cas9, or free gRNA degradation. We have thus demonstrated that these pre-complexed CRISPR RNPs are stable for at least 18 months when stored at –80 °C, and we look forward to re-evaluating this material at the upcoming 24-month time point.

Partnering with GMP gRNA Vendors

The gRNA used to make CRISPR RNPs is either sent directly to Aldevron’s GMP manufacturing facility by the GMP gRNA vendor or it is provided to Aldevron by the client. Because the quality and purity of the gRNA have a significant impact on RNP quantitation and assay performance, we work closely with the leading suppliers of GMP gRNAs and their clients so that they are aware of the influence these factors have on the manufacturing and performance of the final RNP product.

Going forward, Aldevron will remain focused on GMP Cas9 protein and RNP manufacturing, while aligning with the leading GMP gRNA providers to facilitate the transfer of sgRNA to Aldevron in a GMP-compliant manner. We will also ensure that we remain connected to the forefront of gRNA technology through these partnerships.

Providing GMP RNPs

Aldevron has been working to address the needs of the clinical CRISPR space for the last several years, beginning with development of GMP-grade Cas9 products. Our SpyFi™ Cas9 nuclease protein is sold under license of patents and/or patents pending from Integrated DNA Technologies (IDT) and is a new, high-fidelity version of Cas9 with reduced off-targeting available in research, GMP-source, and GMP grades.

With the addition of our RNP complexing and characterization service, Aldevron is filling another gap in the process to create CRISPR material suitable for use in treating humans. Today, we support several clients using CRISPR RNPs to create their cellular therapies. We begin by validating their CRISPR reagents in a small-scale, non-GMP RNP development project, providing the assay results and enough RNP to conduct performance assays in the client’s cell model. Following successful cellular results, the process then moves to a GMP facility, where RNP manufacturing is scaled up and the assays are run under GMP-compliant conditions. The bulk RNP is then transferred to the client or directly to the cell manufacturing facility, where it can be immediately applied or stored for later use. At some point in the future, we envision producing and banking off-the-shelf RNPs for specific client treatments.

Working toward Direct Patient Administration of RNP-Mediated CRISPR Therapeutics

All but one of the genome-editing therapies using CRISPR in the clinic today deliver the CRISPR system ex vivo, where cells in culture are modified with CRISPR to convey a therapeutic benefit prior to being transplanted into a patient. CRISPR RNPs can be delivered to these cells in a variety of ways, with high throughput, clinical-grade electroporation platforms becoming a predominant method because of their efficiency and processivity.

Direct — or in vivo — administration of genome-editing therapies to patients is a much more challenging prospect. It is difficult to control the final destination of RNPs injected into the bloodstream, and targeted delivery to specific cells or tissues in the body presents a major hurdle. Several approaches to in vivo CRISPR genome-editing therapies are focused on diseases originating in the liver, which acts as a sink in its role as filter of the blood. There is significant ongoing development of lipid nanoparticles (LNP) for RNP delivery and a large existing body of data on the use of LNPs for RNA delivery. A reliable LNP solution designed for RNPs has yet to be developed, although there are encouraging results thus far. Other techniques being investigated include cationic lipid formulations,³ poly(glutamic) acid, and modification of the Cas9 protein with a receptor ligand to allow for internalization into specific cell types.⁴ 

Ultimately, the goal is to expand the scope and safety of CRISPR-based therapies to treat the many diseases for which there are few or no alternatives. Before that can happen, however, the technology must be more precise, controlled, and characterized, especially in regard to in vivo genome editing. There is no room for error, and the circumstances demand a well-developed genome-editing technology made with the highest-quality products.

At Aldevron, we are working with our partners — both suppliers and clients — to produce products like CRISPR RNPs that will be used to make therapies we could once only imagine were possible. 

References

1. Genome Editing/Genome Engineering Market by Technology (CRISPR, TALEN, ZFN, Antisense), Product & Service, Application (Cell Line Engineering, Genetic Engineering, Diagnostics, Drug Discovery & Development), End-User and Region – Global Forecast to 2025. Rep. MarketsandMarkets, Jun. 2020. Web. 

2. Gene Editing Market Size Worth Around US$ 9.2 Bn by 2026. Acumen Research and Consulting. 26 Feb. 2020. Web. 

3. Zuris, John A. et al. “Cationic Lipid-Mediated Delivery of Proteins Enables Efficient Protein-Based Genome Editing in Vitro and in Vivo.” Nat Biotechnol. 33: 73-80 (2015). 

4. Rouet, Romain et al. “Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing.” J. Am. Chem. Soc. 140: 6596-6603 (2018).