Home Medizin Ein Durchbruch bei der CRISPR-Bereitstellung verspricht eine sicherere Genbearbeitung

Ein Durchbruch bei der CRISPR-Bereitstellung verspricht eine sicherere Genbearbeitung

von NFI Redaktion

In a recent review published in the journal PNAS, researchers explore non-viral and cell-specific clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) delivery methods and emphasize their advantages for research and gene therapy applications.

Study: Targeted non-viral delivery of genome editors in vivo. Image Source: Catalin Rusnac / Shutterstock.com Study: Targeted non-viral delivery of genome editors in vivo. Image Source: Catalin Rusnac / Shutterstock.com

Enhancing CRISPR-Cas Enzyme Delivery

CRISPR-Cas enzymes offer precision and easy genome editing; however, they are also associated with certain safety concerns as they have the potential to induce permanent changes. The increased specificity of Cas9 represents a leap forward, but targeted delivery remains crucial for minimizing risks.

Viral vectors have been extensively studied as transport vehicles for these enzymes. However, these systems are also associated with the risk of immunogenicity and genetic disturbances.

New alternatives like CRISPR-Cas ribonucleoproteins (RNPs) and messenger RNA (mRNA)-encoded nucleases reduce off-target effects and oncogenic risks, but lack specific targeting. For example, Cas9 RNPs, offering temporary cellular presence and cost-effectiveness, reduce off-target effects and immunogenicity; however, their targeted delivery poses a significant challenge. Hence, there is an urgent need for advanced delivery strategies.

Further research is crucial for developing safer and more precise delivery mechanisms for CRISPR-Cas systems, ensuring targeted genome editing with minimal off-target effects and lower clinical risks.

Ex-vivo Targeted Delivery Methods

Targeted delivery can be achieved through the physical isolation of cells for ex-vivo genome editing. This method is particularly effective for hematopoietic cells, which can be easily isolated and edited outside the body.

Techniques like electroporation have enabled efficient genome editing in T cells as well as hematopoietic stem and progenitor cells (HSPCs), offering a unique potential to revolutionize the treatment of hematologic diseases like sickle cell anemia (SCD) and beta-thalassemia. Despite their effectiveness, a key limitation of electroporation lies in its limited applicability to other tissues as well as potential cytotoxic effects.

Ex-vivo genome editing has also advanced regenerative medicine. For instance, Cas9 RNPs-edited induced pluripotent stem cells (iPSCs) have been used to develop treatments for genetic skin conditions and type 1 diabetes (T1D), demonstrating the potential of this method for replacing or regenerating damaged tissues. This specificity ensures minimal risk of editing unintended cell types and provides a controlled environment for therapeutic interventions.

In vivo Genome Editing: Challenges and Opportunities

In vivo genome editing is limited in its ability to precisely target specific tissues without the advantage of isolation. Techniques like direct injection have achieved local successes in the brain, skin, and tumors, while innovations like cell-penetrating peptides and lipid nanoparticles (LNPs) offer broader delivery possibilities. These advances will inevitably support the development of future CRISPR-Cas systems that can be systemically administered with cell-specific precision.

Advancements in Delivery: LNPs and Encapsulated Delivery Vehicles (EDVs)

LNPs enable systemic delivery and endosomal escape to release genome editing tools directly into cells. LNPs, packing larger genetic sequences and targeting specific tissues, have been explored for their potential in treating complex diseases like familial hypercholesterolemia.

Biologically inspired vehicles, including virus-like particles (VLPs) and extracellular vesicles (EVs), mimic viral delivery mechanisms for targeted editing. These EDVs utilize natural processes for efficient genome editing, showcasing the evolving landscape of CRISPR-Cas delivery methods and their potential to overcome current limitations.

Ex-vivo Precision with VLPs

VLPs have proven promising in delivering tools for precise genome editing for specific cell types in an ex-vivo setting. These engineered particles often use the VSV-G glycoprotein for its broad receptor spectrum, including low-density lipoprotein receptor (LDL-R), to facilitate entry into a variety of human cells such as T cells, B cells, iPSCs, and cluster of differentiation (CD)34+ HSPCs.

This broad applicability is further refined by pseudotyping VLPs with other viral glycoproteins to ultimately target cells based on specific receptor interactions. For example, by utilizing the envelop glycoprotein of human immunodeficiency virus type 1 (HIV-1), VLPs are specifically targeted to CD4+ T cells, increasing delivery specificity and minimizing off-target effects. This approach has been instrumental in reprogramming immune cells for cancer therapy and editing stem cells for regenerative medicine.

In vivo Advances in EDVs

Both VLPs and EVs provide a platform for the direct and localized delivery of CRISPR-Cas9 editors. These EDVs bypass the need for cell-specific targeting when administered directly at the site of interest such as the eye or muscle tissue, offering the possibility to treat diseases like Duchenne muscular dystrophy (DMD) and neurodegenerative disorders. Furthermore, targeted liver editing has been achieved through systemic administration of VLPs to ultimately address liver-associated diseases.

The specificity of these systems is enhanced by displaying targeting molecules on the surface of VLPs, directing the genome editing machinery with minimal off-target activity to specific cells or organs.

Future Directions

The full potential of CRISPR-Cas therapies, especially for non-liver and non-hematologic diseases, hinges on the development of more sophisticated delivery vehicles. The challenge lies in achieving cell-type specificity in vivo without triggering immune reactions or off-target effects.

Recent innovations in LNP formulation and the exploration of biologically inspired EDVs offer promising strategies to facilitate delivery precision. These advances, combined with high-throughput screening and antibody engineering, are likely to lead to the development of minimally invasive and highly specific CRISPR-Cas therapies.

Journal Reference:

  • Tsuchida, CA, Wasko, KM, Hamilton, JR, and Doudna, JA (2024). Targeted non-viral delivery of genome editors in vivo. PNAS. doi:10.1073/pnas.2307796121

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