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CRISPR/Cas Meets AAV: Navigating the Complexities of Gene Editing Delivery

Date: February 2025

The Challenges and Limitations of In Vivo Delivery of CRISPR/Cas Gene Editing Tools

The advent of CRISPR/Cas gene editing has revolutionized molecular biology and biotechnology, offering unparalleled precision in modifying the genome. Initially discovered as part of the bacterial immune system, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and its associated protein Cas serve as a programmable genetic scalpel. By guiding Cas enzymes to specific DNA sequences via RNA templates, researchers can excise, insert, or modify genetic material with remarkable accuracy. The discovery of this mechanism was awarded with the Nobel Price in 2020 and the first therapy utilizing CRISPR/Cas has been approved by the FDA in 2023. 
 

Although some indications benefit from CRISPR-Cas technology through ex vivo applications, the more profound clinical success of CRISPR-Cas therapeutics could be achieved if this genetic tool would be directly administered to patients. However, translating the promise of CRISPR/Cas from ex vivo to in vivo applications, poses significant challenges. These hurdles are particularly pronounced when delivering CRISPR/Cas tools into the body to correct genetic defects, study disease mechanisms, or develop therapeutic interventions. Here, we will explore the primary in vivo delivery strategies, focusing on adeno-associated viruses (AAVs) as delivery vectors, their limitations, and emerging solutions. 

In Vivo Delivery Strategies for CRISPR/Cas

Delivering CRISPR/Cas components into living tissues can be broadly classified into viral and non-viral approaches. 

Viral Vectors: Adeno-associated viruses (AAVs), lentiviruses, and adenoviruses are common vehicles for gene delivery due to their natural ability to infect cells efficiently.

Non-Viral Methods: These include, but are not limited to, lipid nanoparticles (LNPs), polymer-based nanoparticles, gold nanoparticles or exosomes with the advantage of potentially reduced immunogenicity and transient expression of the genetic payload.

Among these, AAV vectors have emerged as a preferred choice for in vivo delivery due to their relatively low immunogenicity, ability to infect both dividing and non-dividing cells, and established safety profile in clinical applications. However, their application for CRISPR/Cas delivery comes with its own set of challenges.

Challenges of AAVs as Delivery Vectors for CRISPR/Cas

1. Limited Cargo Capacity

AAV vectors have a maximum payload capacity of ~4.7 kilobases (kb), which is a significant constraint for CRISPR/Cas systems. For instance, the commonly used Streptococcus pyogenes Cas9 (spCas9) gene alone is ~4.2 kb, leaving little room for the necessary guide RNA (gRNA) and regulatory elements. This restriction often necessitates the use of smaller Cas variants, which may compromise editing efficiency. 

Strategies to overcome the limited packaging capacity include the use of split AAV systems, where components are divided across separate vectors and reassembled inside the co-infected target cells, the exploration of compact Cas9 orthologs or alternative genetically engineered systems, which have smaller genetic footprints.

2. Immune Responses

AAVs can elicit immune responses against both the viral capsid and the transgene, particularly in individuals with pre-existing antibodies to AAV from natural infections. Also, as the most widely utilized Cas proteins are derived from human pathogens, seroprevalence of antibodies against Cas9 is common. Immune activation can reduce therapeutic efficacy and limit repeat dosing. Attenuation of an immune response can be achieved via employing engineered AAV capsids with reduced immunogenicity or novel serotypes to avoid pre-existing immunity. Further possibilities comprise targeting immune privileged organs, such as the eye or the central nervous system, co-administration of immunosuppressive agents to dampen immune responses during treatment or treatment with immunoglobulin-clearing enzymes. In many cases patients are screened for pre-existing antibodies and excluded from treatment if antibody titers are above a defined threshold.  

3. Off-Target Effects and Lack of Precision  

Once CRISPR/Cas is delivered, its activity is not inherently restricted to the target sequence. Off-target edits can lead to unintended genetic modifications with potentially harmful consequences. To overcome this issue researchers are engineering high-fidelity Cas enzymes with reduced off-target activity and designing highly specific gRNAs using advanced bioinformatic tools. Another field of investigation is the incorporation of safety switches, such as drug-inducible systems, to control the temporal and spatial activity of CRISPR/Cas.  

4. Persistent Expression of CRISPR/Cas

Long-term expression of Cas genes delivered by AAV raises concerns about sustained off-target activity and immune responses. Unlike transient delivery methods, AAV-mediated gene editing can persist for longer periods of time, which may not be ideal for therapeutic applications requiring only transient gene editing. Using self-limiting regulatory elements like tissue-specific or inducible promoters to minimize unnecessary expression can reduce some of that risk, as well as engineering transient delivery systems, such as RNA or protein forms of Cas, in conjunction with AAV.  

5. Challenges in Targeting Specific Tissues     

Different AAV serotypes display natural tropism for certain tissues, which may not always align with the intended therapeutic target. Achieving efficient delivery to specific tissues remains challenging. This difficulty can be targeted on different levels. For example, on the treatment administration level by selecting a localized route of administration to limit systemic exposure. On the serotype level, by developing AAV capsids with enhanced tropism for specific tissues through directed evolution, rational design and in silico approaches. On the genetic level using regulatory elements, such as tissue specific promotors and enhancers. On the chemical level, employing targeted delivery strategies such as chemical modification of the capsid or conjugating AAVs with tissue-specific ligands.  

Emerging Solutions and the Future of CRISPR/Cas Delivery

 As research progresses, innovative approaches are addressing many of these challenges. Combinatorial strategies, such as using non-viral vectors like lipid nanoparticles in conjunction with AAVs, are being explored to improve efficiency and reduce side effects. Advances in nanotechnology and synthetic biology are also opening new frontiers for CRISPR/Cas delivery, offering scalable and customizable solutions. 
  
Moreover, regulatory and safety considerations will play a critical role in the broader adoption of in vivo CRISPR/Cas therapies. Robust preclinical testing and the development of standardized delivery protocols are essential for translating these groundbreaking tools into clinical practice.

Conclusion

The application of CRISPR/Cas in vivo promises to transform the landscape of medicine and biology, offering new treatments for previously untreatable diseases. However, the limitations of delivery methods, particularly using AAV vectors, highlight the need for ongoing innovation and multidisciplinary collaboration. As researchers refine these systems, the vision of safe and effective gene editing in living organisms comes closer to reality.

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