In December 2023, the FDA approved the first cell-based gene therapies, Casgevy and Lyfgenia. These two therapies are for treatment of Sickle Cell Disease (SCD), a group of inherited red blood cell disorders. Their success signals ongoing advancement in the field of gene therapy.
Both Casgevy and Lyfgenia are ex vivo treatments. The first step of treatment is the extraction of stem cells from patient bone marrow. Next, these stem cells are modified with gene editing ex vivo. Finally, the modified stem cells are transplanted back in the patient (Figure 1).
Figure 1. Workflows of Casgevy and Lyfgenia.
One of the key technologies of gene therapy is how to deliver gene editing cargo to patients. Currently, electroporation, viral vector and lipid nanoparticles (LNPs) are the three main gene delivery platforms (Table 1).
Table 1. Comparison of electroporation, viral vector and lipid nanoparticle (LNP) gene delivery methods.
Electroporation uses an electric pulse which creates temporary pores to introduce genetic material into cells. Casgevy, the first FDA approved CRISPR therapy, uses electroporation to modify patient stem cells ex vivo. Overall, this method has wide compatibility with different cargo types and sizes. However, electroporation has low cell viability and is limited to ex vivo applications only.
Viral vectors involve a modified virus delivering genetic material to cells. Lyfgenia, another gene therapy approved together with Casgevy, uses a lentiviral vector for ex vivo genetic modification. Viral vectors have the advantage of high cell expression. However, they have limited cargo size potential and carry risk of sustained expression or other safety concerns.
Lipid nanoparticles (LNPs) are widely used as a non-viral vector in gene delivery. Since the COVID-19 pandemic, over 710 million doses of LNP based mRNA vaccines have been administered globally, demonstrating the feasibility and safety of this novel gene delivery method. Benefits include transient expression, limited off-target events, in vivo administration, and scalability for large production.
Besides the success of ex vivo cell-based gene editing therapies like Casgevy and Lyfgenia, more and more in vivo treatments have entered clinical trial phase. For example, NTLA-2001 is a LNP assisted CRISPR/Cas9 therapy for Transthyretin Amyloidosis currently in Phase III clinical trials. Table 2 highlights other recent ongoing in vivo CRISPR clinical trials, many of which use an LNP delivery platform.
Table 2. Recent ongoing In vivo CRISPR Clinical Trials.
The following section will discuss selected cases to demonstrate the features, advantages and challenges of gene editing with LNPs.
NTLA-2001
NTLA-2001 is an in vivo single dose treatment developed by Intellia Therapeutics (Figure 2). NTLA-2001 targets the TTR gene for knockout using CRIPSPR-Cas9 technology. In Phase I, it showed mild adverse events. From its Phase II clinical trial data, NTLA-2001 showed dose dependent reduction of TTR serum concentration from 52% (0.1 mg/kg) to 87% (0.3 mg/kg). NTAL-2001 is the first CRISPR therapy to be administered in vivo to edit genes inside the human body. Currently it is undergoing Phase III clinical trials.
Figure 2. Workflow of NTLA-2001. Source: Intellia Therapeutics.
NTLA-2001 uses LNPs to deliver gene editing material to patients. LP01 is one of the critical components in its lipid formulation. LP01 is an ionizable lipid with a pKa value of 6.1. Its chemical structure is shown below in figure 3.
Figure 3. Chemical structure of LP01 ionizable lipid.
CTX310
CTX310 is an in vivo gene therapy for cardiovascular disease. Utilizing LNPs, CTX310 delivers Cas9 mRNA and guide RNA to the liver to reduce expression of ANGPTL. It is currently undergoing Phase I clinical trials.
CTX310 is developed by CRISPR Therapeutics (Figure 4). For a long time, CRISPR Therapeutics was considered a company focusing on ex vivo therapies (e.g. Casgevy.) More recently though, it expanded its in vivo pipelines to 4 programs. CTX310 and CTX320 are two such therapies targeting cardiovascular disease in Phase I. Two other programs are in the preclinical stage.
Figure 4. Workflow of CTX310.
Source: CRISPR Therapeutics.
EDIT-101
Besides systematic treatments, in vivo gene editing can also be applied locally, such as for inherited retinal diseases (IRDs). EDIT-101 is the pioneer in this field. As early as 2019, Editas initiated a Phase I/II study for treatment of blindness caused by retinal degenerative disorder (Figure 5). EDIT-101 is based on CRISPR technology and uses AAV5 to deliver gene editing material directly to the eyes. Although its safety profile looked promising, EDIT-101's efficacy needed more investigation. In 2022, Editas paused this clinical trial.
Figure 5. Workflow of EDIT-101.
Source: Yan, A. L., et. Vision research, 2023, 206: 108192.
In 2023, a study about LNP-assisted CRISPR genome editing in the mouse retina was published in Nature Communications (Figure 6). It demonstrated that surface modifications of LNPs can alter cellular tropism of mRNA. This enables genome editing in the retina, which can be used to correct genetic mutations that lead to blindness.
Figure 6. A study about LNP-assisted CRISPR genome editing.
Source: Gautam M, et al. Nature communications, 2023, 14(1): 6468.
In summary, as technology matures, genome editing is successfully changing from "bench top" to "bed-side". As delivery methods, electroporation, viral vectors, and LNPs have all been proven effective and include FDA-approved treatments (Figure 7). However, when it comes to treatment regimen, risks, costs, and patient experience, LNP systems suitable for in vivo treatment show significant advantages over other delivery methods that can only apply ex vivo treatments.
Figure 7. Summary of current techniques for gene editing therapies.
Source: Dimitrievska M, et al. Blood Reviews, 2024: 101185.
To develop a successful LNP platform, microfluidic mixing is a robust and efficient tool to synthesis nucleic acid encapsulated LNPs (Figure 8). PreciGenome provides a whole range of lipid nanoparticle synthesis solutions to cover different stages of pipeline development (Figure 9). From early discovery, to pre-clinical testing, to commercial production, PreciGenome's NanoGenerator platform has an offering for you.
Figure 8. Microfluidic mixing for LNP synthesis
Figure 9. Production lines of PreciGenome's NanoGenerator