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CRISPR, Base Editors, and Prime Editors in AAV Gene Therapy

I. Introduction

Gene editing technologies have revolutionized the field of genetic medicine, offering unprecedented opportunities to correct disease-causing mutations at their source. Among these tools, CRISPR-Cas, Base Editors (BEs), and Prime Editors (PEs) stand out as transformative innovations. CRISPR-Cas9, derived from bacterial immune systems, laid the foundation for modern gene editing by enabling targeted DNA double-strand breaks (DSBs). Building on this, Base Editors and Prime Editors have emerged as advanced tools that offer single-nucleotide precision without inducing DSBs, reducing the risks associated with traditional CRISPR systems.

The therapeutic potential of these gene editing tools is immense, particularly for treating genetic disorders, cancers, and infectious diseases. However, their success hinges on the development of efficient delivery systems capable of safely and precisely delivering these tools to target cells. This article explores the mechanisms, innovations, and challenges of CRISPR, Base Editors, and Prime Editors in gene therapy, with a focus on delivery strategies, real-world applications, and the biotech companies leading the charge in this field.

 

II. CRISPR-Cas Systems

a. CRISPR-Cas9

The mechanism of the CRISPR-Cas9 system involves using a guide RNA (gRNA) to direct the Cas9 nuclease to a specific DNA sequence, where it introduces a DSB. The cell repairs the break through Non-Homologous End Joining (NHEJ), which often results in insertions or deletions (indels) leading to gene disruption, or Homology-Directed Repair (HDR), which allows precise gene correction using a donor template. This system is widely used for gene knockout, gene correction, gene insertion, and transcriptional regulation, offering high efficiency, versatility, and ease of use. However, it carries the risk of off-target effects and unintended mutations due to DSBs. Several companies are advancing CRISPR-Cas9-based therapies: CRISPR Therapeutics is developing CTX001 for sickle cell disease and beta-thalassemia, with promising clinical trial results; Editas Medicine is advancing EDIT-101 for Leber congenital amaurosis, a genetic blindness disorder; and Intellia Therapeutics is working on NTLA-2001 for transthyretin amyloidosis, which has shown positive Phase 1 clinical trial results. The success of these therapies often relies on robust viral vector manufacturing and AAV production service capabilities.

 

b. CRISPR-Cas12 (Cpf1)

CRISPR-Cas12, similar to Cas9, creates staggered cuts (sticky ends) instead of blunt ends, which can improve HDR efficiency. This system is used for gene editing and diagnostics, such as the DETECTR platform for rapid viral detection. One of its key advantages is its smaller size compared to Cas9, which makes it easier to package into delivery vectors like adeno-associated virus (AAV), and it has shown reduced off-target effects in some cases. However, it is less widely adopted than Cas9, limiting its current applications. Mammoth Biosciences is a leading company in this space, developing CRISPR-Cas12-based diagnostics, including the DETECTR platform, which has been used for rapid and accurate detection of viral pathogens.

 

c. CRISPR-Cas13

CRISPR-Cas13 is a unique gene editing tool that targets RNA instead of DNA, making it particularly useful for RNA editing, viral RNA targeting, and diagnostics, such as the SHERLOCK platform for RNA detection. One of its key advantages is that it poses no risk of genomic DNA damage, as it operates exclusively at the RNA level. However, its applications are limited to RNA, which restricts its use compared to DNA-targeting systems like CRISPR-Cas9. Sherlock Biosciences is a prominent company leveraging CRISPR-Cas13 technology, developing the SHERLOCK diagnostics platform for rapid and accurate detection of RNA, including applications for COVID-19 and other infectious diseases. The scalable AAV production and AAV packaging service for such applications are crucial.

 

d. CRISPR-CasΦ (Cas12f)

CRISPR-CasΦ (Cas12f) is an ultra-compact Cas protein, measuring less than half the size of Cas9, which allows it to be packaged into smaller delivery vectors like AAV. This makes it particularly useful for gene editing in space-constrained delivery systems, where larger Cas proteins may not fit. Its small size and efficient editing capabilities are significant advantages, though it is still in the early stages of development, limiting its current applications. Stella Therapeutics is one of the companies exploring the potential of CasΦ for gene therapy applications, with preclinical studies currently underway to evaluate its efficacy and safety.

 

III. Base Editors: Precision Without DSBs

Base Editors are engineered fusion proteins that combine a catalytically impaired Cas9 (nickase) with a deaminase enzyme, enabling precise single-nucleotide changes without inducing DSBs. There are two main types: Cytosine Base Editors (CBEs), which convert C•G to T•A, and Adenine Base Editors (ABEs), which convert A•T to G•C. These tools are particularly useful for correcting point mutations associated with genetic diseases, such as sickle cell anemia and progeria. A key advantage of Base Editors is their reduced risk of indels and off-target effects compared to traditional CRISPR-Cas9 systems. Several companies are actively working on Base Editor technologies, including Beam Therapeutics, which is developing BEAM-101 for sickle cell disease and BEAM-201 for cancer immunotherapy, and Verve Therapeutics, which is advancing VERVE-101 for familial hypercholesterolemia. These companies are making significant progress in preclinical and clinical studies, demonstrating the potential of Base Editors for precise and safe gene editing.

 

IV. Prime Editors: Versatile and Precise

Prime Editors utilize a Cas9 nickase fused to a reverse transcriptase and a prime editing guide RNA (pegRNA). The pegRNA directs the editor to the target DNA site and provides a template for precise DNA editing, enabling insertions, deletions, and base substitutions with high precision and minimal off-target effects. This system offers broader editing capabilities compared to Base Editors, making it suitable for correcting a wide range of genetic mutations, including those that are challenging to address with other gene editing tools. Companies like Prime Medicine are at the forefront of developing Prime Editor technologies, focusing on applications for genetic diseases such as Duchenne muscular dystrophy and cystic fibrosis. The successful development of these therapies depends heavily on efficient AAV manufacturing and the provision of GMP AAV products from reliable AAV CDMOs.

 

V. Choosing the Right Delivery Vector

Selecting the appropriate delivery vector for gene editing tools depends on several critical factors. First, cargo size is a key consideration, as advanced editors like Prime Editors and Base Editors require larger delivery capacity compared to the more compact CRISPR-Cas9. This makes it challenging to package these editors into smaller vectors like AAV, often necessitating the use of dual-vector systems or alternative delivery methods. This is where advanced AAV packaging strategies and AAV production service providers come into play.

Second, target tissue specificity plays a significant role; for example, AAV vectors are ideal for tissue-specific delivery due to their natural tropism for certain organs, while lipid nanoparticles (LNPs) offer greater flexibility for systemic applications, such as targeting the liver or immune cells. Custom AAV production and the availability of diverse AAV libraries are crucial for achieving this specificity.

Third, the safety profile of the delivery system is crucial, as minimizing immune responses and off-target effects is essential for clinical success. This demands rigorous quality control throughout AAV manufacturing, from plasmid preparation and plasmid production (including GMP plasmid manufacturing) to final AAV preparation and GMP manufacturing. Reliable plasmid CDMOs and plasmid service providers are essential partners here.

Finally, the choice between ex vivo vs. in vivo applications must be considered. Ex vivo editing, such as modifying CAR-T cells in the lab before reinfusion, allows for precise control over the editing process, while in vivo delivery requires robust and safe viral vector production that can efficiently deliver gene editing components to target tissues without causing adverse effects. Balancing these factors is essential to ensure the efficacy, safety, and success of gene editing therapies, underscoring the vital role of specialized AAV manufacturing service providers in this rapidly evolving field.

 

References

1. Anzalone, A. V., et al. (2019). “Search-and-replace genome editing without double-strand breaks or donor DNA.” Nature, 576(7785), 149-157.
2. Gaudelli, N. M., et al. (2017). “Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage.” Nature, 551(7681), 464-471.
3. Doudna, J. A., & Charpentier, E. (2014). “The new frontier of genome engineering with CRISPR-Cas9.” Science, 346(6213), 1258096.
4. Anzalone, A.V., et al. (2020). Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol, 38, 824–844.

AAV-Cas13d for Multiplexed Immunosuppressive Gene Inhibition in Cancer Therapy

Feifei Zhang et al. recently published a research article in Nature Biotechnology titled “Multiplexed inhibition of immunosuppressive genes with Cas13d for combinatorial cancer immunotherapy.” This paper describes a novel multiplexed, universal combinatorial immunotherapy approach using CRISPR-Cas13d to silence multiple endogenous immunosuppressive genes within the tumor microenvironment (TME), thereby promoting TME remodeling and enhancing antitumor immunity.

 

Background and Rationale

Cancer cells exploit the immunosuppressive TME to evade immune surveillance and resist therapeutic interventions. Immune checkpoint blockade (ICB) therapies, such as anti-PD-1/PD-L1 and anti-CTLA4 antibodies, have revolutionized cancer treatment by restoring T-cell-mediated immunity. However, the clinical success of ICB is limited, as many patients exhibit either intrinsic or acquired resistance, with single-agent therapies often insufficient to counteract the complexity of the TME. This limitation has driven the pursuit of combination therapies that can simultaneously target multiple immunoregulatory pathways. Conventional approaches to combinatorial immunotherapy face challenges, such as drug toxicity and regulatory complexity. The advent of CRISPR-Cas systems has introduced new possibilities for precise gene regulation. Cas13d, a smaller RNA-targeting member of the CRISPR family, enables simultaneous knockdown of multiple genes at the RNA level, offering an innovative approach to TME remodeling without introducing permanent changes to the genome. The rationale behind this study is grounded in the understanding that individual immunosuppressive genes, such as PD-L1, Galectin-9, Galectin-3, and CD47, play pivotal roles in maintaining the immunosuppressive nature of the TME. Targeting these genes in combination is hypothesized to generate synergistic antitumor effects by disrupting multiple layers of immune suppression. The authors proposed the Multiplex Universal Combinatorial Immunotherapy via Gene silencing (MUCIG) platform, which utilizes Cas13d to silence these genes at the RNA level.

 

Methods

Gene Selection and Pool Design:
From an initial list of 588 immunosuppressive genes identified via databases and literature, 19 targets were prioritized, with four—Cd274, Lgals9, Lgals3, and Cd47 (PGGC pool)—selected for their immune-modulating roles and consistent tumor expression.

CRISPR-Cas13d System Design:
Cas13d was chosen for its compact size, allowing a single AAV to deliver both the protein and gRNAs. Its RNA-targeting mechanism avoids DNA breaks, enhancing safety. Efficient gRNAs were computationally optimized and tested before finalizing.

AAV Vector Design:
An all-in-one AAV vector was engineered with U6 and EFS promoters and mutant direct repeats to enhance knockdown efficiency. AAV9 serotype was used for its ability to transduce tumor and immune cells. This highlights the importance of robust AAV construction and AAV capsid engineering.

Animal Models:
Syngeneic murine tumor models—E0771 (breast), Colon26 (colon), B16F10 (melanoma), Pan02 (pancreatic)—were used. AAV-Cas13d-PGGC was administered intratumorally, typically on days 5, 9, and 14.

Combination Therapy:
Combining AAV-Cas13d-PGGC with anti-GR1 antibodies enhanced efficacy by depleting MDSCs and neutrophils. Additional assays confirmed increased MHC-I expression, T-cell activation, and tumor cell susceptibility to T-cell-mediated killing.

 

Results and Key Data

1. Gene Silencing Efficiency:
Cas13d efficiently silenced the four target genes both at the RNA and protein levels, which displayed more significant efficiency compared to shRNA methods. Knockdown was specific, with minimal off-target effects, as confirmed by transcriptomic profiling. Enhanced knockdown of specific targets was observed with optimized direct repeats (Mut-DR). This method is effective across various syngeneic tumor models, showcasing broad efficacy in TME modulation, supporting its potential as a custom AAV production strategy.

2. Antitumor Efficacy:
AAV-Cas13d-PGGC significantly reduced tumor growth in multiple cancer models, including checkpoint-resistant E0771 breast cancer, Colon26 colon cancer, B16F10 melanoma, and immunologically “cold” Pan02 pancreatic cancer (Figure 1). Both B16F10 and Pan02 models are typically resistant to traditional ICB therapies, making these results particularly noteworthy. Tumor reduction correlated with improved immune cell infiltration and activity.

Figure-1.-A-four-gene-combination-immunotherapy-AAV–Cas13d–PGGC-demonstrates-broad-antitumor-activity-across-diverse-syngeneic-cancer-models.

Figure 1. A four-gene combination immunotherapy AAV–Cas13d–PGGC demonstrates broad antitumor activity across diverse syngeneic cancer models.

 

3. TME Remodeling:
AAV–Cas13d–PGGC therapy reshaped the immune landscape within tumors. Here are the observations:
Increased T cells: CD8+ and CD4+ T cells infiltrated tumors treated with AAV-Cas13d-PGGC.
Reduced suppressive populations: Profiling revealed downregulation of key immunosuppressive genes in the treated groups. Neutrophils and myeloid-derived suppressor cells (MDSCs) were depleted in treated tumors (Figure 2). Notably, specific pathways like neutrophil chemotaxis were consistently affected, aligning with the observed phenotypic changes.
Enhanced immune signaling: These changes correlated with enhanced production of IFN-γ by T cells, increased expression of MHC-I on tumor cells, and improved antigen presentation, indicating a shift toward a proinflammatory and antitumor TME.

  • Safety and Specificity:
    Treatment with a high-fidelity version of Cas13d (hfCas13d) maintained strong antitumor activity without causing systemic toxicity or weight loss. Minimal off-target effects were observed, confirmed through mRNA-seq analysis. No significant toxicity, such as weight loss or liver damage, was reported in treated models.

  • Combination Strategies:
    Combining AAV-Cas13d-PGGC with anti-GR1 antibodies to deplete both neutrophils and MDSCs enhanced tumor reduction compared to monotherapy.

 

4. Comparison with Alternative Methods

  • Versus Cas9:
    o Cas13d exhibited better transduction efficiency and efficacy without the genomic instability caused by DNA breaks from Cas9.

  • Versus shRNA:
    o Cas13d-mediated knockdown achieved stronger antitumor effects despite similar in vitro gene silencing efficiency.

 

5. Discovery of Synergistic Mechanisms

  • Immunogenicity:
    o Cas13d increased IFN-γ expression in CD8+ T cells, enhancing immune activation and antigen presentation.
    o MHC-I expression was upregulated, improving T-cell recognition and tumor cell killing.

  • Direct vs. Indirect Effects:
    oTumor growth reduction was largely immune-mediated, as shown by diminished efficacy in immune-deficient models.

Figure-2.-AAV–Cas13d–PGGC-treatment-remodels-the-immunosuppressive-TME.

Figure 2. AAV–Cas13d–PGGC treatment remodels the immunosuppressive TME.

 

Conclusion

The study establishes the MUCIG platform as a promising strategy for combinatorial cancer immunotherapy. By leveraging the RNA-targeting capabilities of Cas13d, the platform simultaneously disrupts multiple immunosuppressive pathways in the TME, leading to significant antitumor responses across diverse tumor models. The use of Cas13d, which avoids permanent DNA modifications, adds a layer of safety and reversibility, making it particularly attractive for clinical translation. The new discovery of the PGGC pool’s broad efficacy in checkpoint-resistant cancers represents a significant advance in immunotherapy. Moreover, traditional approaches, such as monoclonal antibodies and small molecules, often focus on single pathways, which can lead to incomplete responses or therapeutic resistance. The advent of RNA-targeting tools like Cas13d represents a paradigm shift, enabling precise and flexible modulation of multiple genes. This aligns with the growing interest in immunogenomics and personalized medicine, where therapies are tailored to the specific molecular and immunological profiles of individual tumors.

This groundbreaking research underscores the need for robust AAV production capabilities and specialized AAV services. Companies offering comprehensive solutions like AAV packaging service, plasmid preparation, plasmid manufacturing, and GMP plasmid manufacturing are crucial. As a leading AAV CRO and CDMO, such as PackGene, providing GMP AAV and custom AAV options, along with AAV manufacturing service and AAV construction service, will be vital to translate such innovative research into therapies. Expertise in viral vector production, plasmid production service, and ensuring GMP plasmids will accelerate the development of future AAV libraries and AAV products to meet patient needs. The study highlights the expanding potential of AAV technology in cancer immunotherapy, pushing the boundaries of vector manufacturing and AAV manufacturing.

 

Reference:
1. Multiplexed inhibition of immunosuppressive genes with Cas13d for combinatorial cancer immunotherapy.
Zhang F, Chow RD, He E, Dong C, Xin S, Mirza D, Feng Y, Tian X, Verma N, Majety M, Zhang Y, Wang G, Chen S.
Nat Biotechnol. 2025 Jan 16.

Advances in AAV-SB Transposon Hybrid Systems for Liver-Targeted Gene Therapies

AAV-SB-Transposon-Hybrid-Systems

*Nicolás Sandoval-Villegas, Zoltán Ivics, The best of both worlds: AAV-mediated gene transfer empowered by LNP delivery of Sleeping Beauty transposase for durable transgene expression in vivo, Molecular Therapy, Volume 32, Issue 10, 2024, Pages 3211-3214, ISSN 1525-0016, https://doi.org/10.1016/j.ymthe.2024.09.002.
(https://www.sciencedirect.com/science/article/pii/S1525001624005896)

Liver-targeted gene therapies are at the forefront of treating genetic diseases, particularly with the development of adeno-associated virus (AAV) and Sleeping Beauty (SB) transposon systems. These technologies offer a range of benefits for both pediatric and adult patients, including efficient and stable genome integration. The SB transposon system has demonstrated a genome-wide integration profile, making it an attractive tool for gene therapies. When combined with AAV vectors, it enhances the potential for precise and stable gene delivery, particularly for liver-targeted applications.

AAV vectors are widely used in gene therapy due to their relatively low immunogenicity and ability to target specific tissues, such as the liver. However, AAV vectors are limited by their cargo capacity, which can restrict the size of the therapeutic gene they can carry. To overcome this limitation, hybrid vector systems have been developed, combining the advantages of AAV with the genomic integration capabilities of the SB transposon system.

Recent studies have explored alternative delivery methods for the SB transposon system, including the use of polyethylenimine (PEI) to form DNA complexes for intravenous injection, targeting the lungs. Physical delivery methods like hydrodynamic injection have also been employed for liver-targeted gene delivery, ensuring efficient transposon transfer.

The most promising advancement in this field is the development of AAV-SB hybrid vectors. These hybrid systems take advantage of the natural ability of viruses to penetrate cell membranes while enabling stable genomic integration via the SB transposon system. Zakas et al. described a hybrid vector approach incorporating SB transposon components into various viral vectors, including integrase-defective lentiviral particles, adenovirus vectors, herpes simplex virus vectors, and baculovirus vectors. This combination allows for the efficient transfer of large genetic payloads into target cells.

One innovative application of these hybrid vectors is in the targeting of hematopoietic stem cells (HSCs) in vivo. Researchers have demonstrated that autologous HSCs can be mobilized into peripheral blood and genetically engineered using a hybrid adenovirus/SB transposon vector system. This approach has led to functional, genetically modified HSCs in humanized mouse models, showing great potential for future clinical applications.

AAV vectors, although limited in cargo capacity compared to adenoviral vectors, remain a critical tool in gene therapy due to their well-established safety profile and their ability to achieve tissue-specific delivery. Hybrid AAV-SB vectors offer a solution to the cargo limitations of AAV, while still benefiting from the transposon’s ability to mediate efficient genomic integration. In contrast, other methods, such as CRISPR-Cas9, rely on double-strand DNA breaks and repair mechanisms, which carry risks such as off-target effects and chromosomal translocations.

The safety of AAV-SB hybrid systems, particularly in terms of genomic integration, remains a topic of ongoing research. While the SB transposon system is considered to have a random integration profile, this randomness may reduce the risk of insertional mutagenesis compared to retroviral or lentiviral vectors, which often integrate near active genes. However, careful evaluation of the genomic integration sites is required to ensure long-term safety in clinical applications.

The use of AAV-SB transposon hybrid systems for liver-targeted gene therapies represents a promising advancement in the field of genetic medicine. These hybrid systems combine the safety and targeting advantages of AAV with the stable integration capabilities of the SB transposon system, potentially providing new treatment options for genetic liver diseases. Future research will focus on optimizing the delivery, safety, and efficacy of these hybrid vectors to ensure their successful transition from experimental models to clinical therapies.

Another promising approach for gene editing applications is PackGene’s SB-100 mRNA system. SB-100 mRNA offers an efficient and non-viral method for delivering the Sleeping Beauty transposon system into target cells, facilitating precise gene integration without the need for viral vectors. This method enhances safety by eliminating concerns related to viral vector immunogenicity, while maintaining the high efficiency of transposon-mediated integration. PackGene’s SB-100 mRNA is especially valuable for gene editing in liver-targeted therapies, as it can deliver larger genetic payloads, circumventing the cargo limitations associated with AAV vectors. As part of PackGene’s expanding mRNA service portfolio, this system represents a cutting-edge tool for advancing both preclinical and clinical gene editing applications.

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Novel Approach in T Cell Engineering: Lipid Nanoparticles Enable Advanced Genome Editing for Cancer Therapies

Revolutionizing CAR T Cell Therapy with Lipid Nanoparticles

Chimeric antigen receptor (CAR) T cell therapy has transformed cancer treatment by turning a patient’s own T cells into powerful cancer-fighting agents. However, as the technology advances, there is an increasing need for more sophisticated genetic modifications. The latest breakthrough involves using lipid nanoparticles (LNPs) to deliver CRISPR-Cas9 genome editing tools to human primary T cells, a method that could enhance CAR T cell therapies and overcome current limitations.

Gene-editing-with-lipid-nanoparticles-in-human-primary-T-cells

Fig. Gene editing with lipid nanoparticles in human primary T cells.

Challenges with Current T Cell Engineering Methods

Traditional methods of T cell engineering involve using viral vectors or electroporation to deliver genetic material into cells. While effective, these approaches have notable drawbacks. Viral vectors can provoke immune responses, have limited capacity, and are costly to produce. Electroporation, on the other hand, uses electrical pulses to introduce genetic material into cells but can compromise cell viability, especially during complex, multi-step engineering processes.

 

Why Lipid Nanoparticles?

LNPs offer a promising alternative for T cell engineering. These synthetic particles encapsulate and protect RNA, delivering it into cells in a way that resembles the natural uptake of low-density lipoproteins (LDL). This gentle and efficient method avoids the harsh conditions of electroporation, maintaining high cell viability while enabling complex gene editing and protein expression.

 

The GenVoy-ILM™ T Cell Kit for mRNA: A Game Changer

In a recent study, researchers showcased a novel method using the GenVoy-ILM™ T Cell Kit for mRNA to edit T cells via LNPs. The team demonstrated the sequential delivery of Cas9 mRNA and single-guide RNA (sgRNA) to knock out the T cell receptor (TCRαβ), a step toward creating universal CAR T cells from allogeneic donors. This multi-step approach also included introducing CAR mRNA to generate CAR T cells capable of targeting cancer cells.

 

How It Works: LNP-Mediated CRISPR-Cas9 Editing

LNPs encapsulate Cas9 mRNA and sgRNA, which guide the Cas9 protein to the target DNA within T cells, inducing double-strand breaks that are repaired by the cell, often resulting in gene knockouts. This process disrupts inhibitory pathways exploited by the tumor microenvironment, enhancing the effectiveness of CAR T cells. The researchers achieved a knockout efficiency of 80% with high cell viability, outperforming traditional methods.

 

Potential Impact on CAR T Cell Therapy

Using LNPs for T cell engineering could significantly enhance the production of CAR T cells by improving cell yield and viability, reducing manufacturing costs, and streamlining the production process. This method is also scalable, making it suitable for clinical applications. The study highlighted that LNP-engineered CAR T cells maintained their therapeutic potential, effectively killing cancer cells in co-culture assays.

 

A Step Towards the Future of Cell Therapies

The success of LNPs in genome editing and protein expression, as demonstrated in this study, represents a significant advancement in T cell engineering. By leveraging a clinically relevant, scalable method that maintains cell viability and functionality, LNPs could accelerate the development of next-generation CAR T cell therapies. This approach not only addresses current challenges but also sets the stage for more complex and personalized cell-based treatments in the future.

As the field of cell and gene therapy continues to evolve, innovative delivery systems like LNPs will be crucial in overcoming the limitations of existing technologies. With the potential to make gene editing safer, more efficient, and more accessible, LNPs are poised to play a transformative role in the future of cancer treatment and beyond.

Source: https://www.bioprocessonline.com/doc/genome-editing-of-human-primary-t-cells-with-lipid-nanoparticles-0001

New Lipid Nanoparticles Deliver CRISPR-Cas9 to Knock Down Angptl3 in Mice

A team of researchers has developed a new way to deliver CRISPR-Cas9 genome editing tools directly to the liver, targeting a gene called Angptl3 that’s linked to high cholesterol and triglyceride levels. This breakthrough could pave the way for new treatments for lipoprotein metabolism disorders, which are a major risk factor for heart disease.

 

Why Target Angptl3?

Angptl3, or angiopoietin-like 3, is an enzyme that regulates levels of fats in the blood. Some people naturally have mutations in the Angptl3 gene that cause it to stop working, leading to lower levels of LDL cholesterol and triglycerides without any apparent health issues. Because of this, Angptl3 has become a hot target for new cholesterol-lowering therapies. Current treatments include monoclonal antibodies and antisense oligonucleotides (ASOs), which have shown promise in clinical trials, but these approaches are short-lived and need frequent doses.

 

The Promise of CRISPR-Cas9

CRISPR-Cas9 offers a more permanent solution by directly editing genes in the body. The Cas9 enzyme acts like molecular scissors, cutting DNA at precise locations guided by RNA molecules. Once the DNA is cut, the cell’s natural repair mechanisms kick in, often disabling the targeted gene. However, getting CRISPR components safely and efficiently into the right cells has been a major hurdle. Viral vectors have been used but come with risks like unintended genetic mutations and immune reactions.

 

Lipid Nanoparticles to the Rescue

Enter lipid nanoparticles (LNPs), tiny fat-like particles that can carry RNA and other molecules into cells. LNPs are already used in some FDA-approved drugs for delivering siRNA, a technology similar to CRISPR but less permanent. Researchers have now developed a new type of LNP that delivers Cas9 mRNA and a guide RNA targeting Angptl3, directly to liver cells in mice.

The new LNP, known as 306-O12B, was shown to be significantly more efficient than the FDA-approved MC-3 LNP, which is considered the gold standard for delivering nucleic acids to the liver. In tests with wild-type C57BL/6 mice, the LNP system knocked down the Angptl3 gene in the liver, leading to substantial reductions in serum levels of ANGPTL3 protein, LDL cholesterol, and triglycerides.

 

Long-Lasting Effects Without Side Effects

One of the standout findings of this study is the durability of the gene editing effects. A single dose of the LNP-CRISPR system maintained its therapeutic impact for at least 100 days, far longer than current antibody or ASO treatments. Importantly, no off-target effects were detected at the top nine predicted sites, and there were no signs of liver toxicity, making this approach both effective and safe in the tested mice.

 

What This Means for the Future

This study highlights the potential of using LNPs for delivering CRISPR-Cas9 in a safe and targeted way, specifically for treating disorders like hyperlipidemia that have a clear genetic component. By offering a more permanent fix compared to traditional therapies, this method could reduce the need for frequent treatments and improve patient outcomes.

While more research is needed, especially in larger animals and eventually humans, the success of this LNP system in mice is a promising step toward the clinical use of CRISPR-based therapies. If these findings hold up in further studies, we could be looking at a new frontier in the fight against heart disease and other conditions linked to high blood lipid levels.

This innovative approach not only makes genome editing safer and more precise but also underscores the growing role of nonviral delivery methods like LNPs in advancing gene therapy. As the technology continues to evolve, it brings us closer to the goal of making gene therapy accessible and affordable for everyone.

This discovery is yet another exciting chapter in the ongoing story of CRISPR, showing just how far we’ve come—and how much potential still lies ahead—in the quest to edit our way to better health.

Source: https://www.pnas.org/doi/full/10.1073/pnas.2020401118

AAV CRISPR/Cas9 Gene Editing: A New Horizon in Huntington’s Disease Treatment

Recent advancements in gene therapy have unveiled new possibilities for treating Huntington’s Disease (HD), a neurodegenerative disorder characterized by the progressive decline of motor and cognitive functions. This condition is caused by the expansion of CAG repeats in the huntingtin (HTT) gene, leading to the production of a toxic mutant huntingtin (mHTT) protein. The relentless progression of HD has driven scientists to explore innovative therapeutic strategies, with CRISPR/Cas9 gene editing emerging as a promising contender.

In a recent study, researchers investigated the potential of CRISPR/Cas9 to treat HD by targeting the mutant allele directly in a large animal model that closely mirrors human physiology. The study utilized genetically engineered HD knock-in pigs, which were chosen for their anatomical and physiological similarities to humans, particularly in brain structure and function. This choice of model is significant, as it provides a more accurate representation of the disease’s impact and the therapeutic outcomes in a context that closely resembles human conditions.

The research team employed an adeno-associated virus (AAV) vector, provided by PackGene, to deliver the CRISPR/Cas9 system. This vector carried the Cas9 enzyme, a single-guide RNA (sgRNA) designed to target the HTT gene, and donor DNA containing the normal CAG repeat sequence. The treatment was administered through either intracranial or intravenous injections, aiming to assess the impact on mHTT expression and the resulting neurological effects.

The findings from this study were remarkable. A single injection of the AAV-CRISPR/Cas9 construct led to a significant reduction in the expression of the mutant HTT protein in the treated pigs. This reduction was accompanied by notable improvements in neurological symptoms, including motor function, and a substantial decrease in neurodegeneration in brain regions most affected by HD. These results provide strong evidence that CRISPR/Cas9 can effectively mitigate the pathological effects of HD at the molecular level, offering a potential therapeutic avenue that could alter the course of the disease.

One of the key strengths of this study lies in its use of pigs as the model organism. Unlike smaller animals such as mice, pigs share several critical physiological traits with humans, making them an ideal model for preclinical testing of gene-editing therapies. The successful application of CRISPR/Cas9 in this large animal model not only validates the approach but also underscores its potential for translation into clinical applications for human patients.

However, while the study’s outcomes are encouraging, they also highlight some of the challenges that remain. The efficiency of gene replacement was lower than anticipated, which is a known limitation in the application of CRISPR/Cas9, particularly in large animals. Additionally, the potential for off-target effects, though not observed at significant levels in this study, continues to be a concern that warrants further investigation. The study also opens up possibilities for exploring alternative delivery methods, such as non-viral vectors like lipid nanoparticles, which could enhance the efficiency and safety of gene therapy for neurological disorders.

The involvement of PackGene in providing the AAV vector for this research was crucial to its success, demonstrating the importance of collaborative efforts in advancing the field of gene therapy. This study is a significant step forward in the development of CRISPR/Cas9 as a viable treatment for Huntington’s Disease and potentially other neurodegenerative disorders caused by genetic mutations. As researchers continue to refine this technology, the prospect of using CRISPR/Cas9 to offer lasting, effective treatment for HD becomes increasingly attainable, bringing new hope to those affected by this debilitating disease.

Source: https://www.packgene.com/frontier/107-2/

Advancing Miniature Gene Editing: High-Efficiency IscB Variants Enable In Vivo Therapeutic Potential

Over the past decade, gene editing technologies, particularly CRISPR/Cas9 and its derivatives, have rapidly advanced, enabling more precise and efficient genetic modifications. This progress offers new possibilities for better understanding life processes and developing precise gene therapies. The first ex vivo gene editing therapy based on CRISPR/Cas9, Casgevy, was approved at the end of 2023 for the treatment of thalassemia and sickle cell anemia1. In vivo gene therapies, delivered via liver-targeted LNP-mRNA, have also shown promising clinical trial data. Gene editing has thus entered the era of clinical applications. However, there are still significant challenges in targeting organs other than the liver for in vivo gene editing. Currently, the main vectors for gene delivery beyond the liver are adeno-associated viruses (AAVs), but their packaging limit of about 4.7 kb makes it difficult to deliver nucleases like Cas9 and its derivatives. Therefore, finding small, efficient gene editing systems is crucial for safe and effective in vivo delivery.

In recent years, several small Cas proteins have been reported, including the miniature Cas12f proteins2-4, their evolutionary ancestor TnpB5, 6, and eukaryotic homologs Fanzor7, 8. However, because the Cas12f family has only one RuvC domain, it is challenging to use them for derived technologies like base editing and prime editing, which rely on nickase enzymes.

In 2021, Feng Zhang’s team discovered the IscB nuclease, encoded by the IS200/IS605 transposon superfamily, through targeted mining and analysis of sequencing data9. IscB, a possible evolutionary ancestor of Cas9, generally has only about 400-500 amino acids (approximately one-third the length of SpCas9) and uses a non-coding RNA (ωRNA) to guide the protein to recognize and cut DNA. This makes IscB a potential candidate for creating a nickase enzyme that can be fused with cytidine deaminase (APOBEC), adenosine deaminase (TadA), or reverse transcriptase (RT) to construct miniaturized base editors (BE) or prime editors (PE) that can be fully packaged into a single AAV, offering significant clinical application potential. However, IscB’s activity in mammalian cells is very limited. For example, the editing efficiency of OgeuIscB in HEK293FT cells is less than 5%. Thus, enhancing IscB’s gene editing activity to levels comparable to Cas9 is a primary challenge.

On August 2, 2024, Dali Li’s team from East China Normal University published a study in Molecular Cell titled “Engineering IscB to Develop Highly Efficient Miniature Editing Tools in Mammalian Cells and Embryos.” The research combines protein engineering, RNA structure optimization, and embryo injection technologies to successfully obtain IscB variants (eIscB-D) with ultra-high editing activity in human and mouse-derived cell lines. By fusing IscB nickase (eIscBH339A) with adenosine deaminase (TadA-8e) or cytidine deaminase (hA3A*), they developed highly active miniature single-base editors eiABE and eiCBE. This system showed efficient in vivo editing activity in mouse embryos and can be used to construct animal models of diseases such as albinism.

To enhance IscB protein activity, researchers introduced arginine mutations at key positions based on rational design10, 11. After three rounds of iterative screening, they obtained an enhanced IscB (named eIscB) with editing efficiency up to 22.4 times higher than wild-type IscB, with an average improvement of 7.5 times. Additionally, by fusing a non-sequence-specific DNA double-strand binding protein (HMG-D), they increased IscB’s affinity for target DNA, achieving a maximum editing efficiency of 91.3% with high-activity eIscB (eIscB-D). They further optimized the guide RNA, obtaining a highly efficient ωRNA (named eωRNA), which is about 20% shorter than the wild-type ωRNA, reducing the difficulty of industrial synthesis. The optimized eIscB-D/eωRNA system achieved an average editing efficiency improvement of 20.2 times compared to the original IscB/ωRNA.

Highly-efficient-miniature-editing-tools-in-mammalian-cells-and-embryos

Credit: Molecular Cell

By introducing alanine mutations at key catalytic sites of the RuvC domain and screening, the researchers developed IscB nickase (eIscBH339A), and fused it with adenosine deaminase (TadA-8e) and cytidine deaminase (hA3A*) to create highly active miniature single-base editors eiABE and eiCBE, with maximum editing efficiencies of 73.6% and 79.2%, respectively.

Previously, there was no evidence that IscB could produce efficient editing in mice. The researchers first screened targets for the PCSK9 and Tyr genes in the mouse N2a cell line. Sequencing results showed that eIscB-D achieved 58% editing efficiency at the PCSK9-sg29 target and 47.1% at the Tyr-sg21 target. They then injected the eIscB-D/eωRNA system targeting exon 1 of the Tyr gene into mouse embryos, disrupting the expression of the albinism gene, and successfully created a mouse model of albinism. In the F0 generation, 75% (9/12) of the mutant mice showed high-efficiency editing (average editing efficiency of 58.8%), with 5 mice exhibiting nearly 100% editing, resulting in a completely albino phenotype. This study demonstrated for the first time that eIscB-D can produce efficient editing in mouse-derived cell lines and can efficiently create disease animal models through embryo injection.

In summary, this study successfully developed highly active miniature gene editing tools based on IscB, achieving efficient in vivo editing in mice for the first time. This research significantly increases the potential for safe and efficient delivery of base editing or prime editing systems using a single AAV vector, expands the application scenarios of gene editing tools, and provides efficient candidate technologies for future in vivo gene therapy.

 

Reference:
1. Wong, C. (2023). UK first to approve CRISPR treatment for diseases: what you need to know. Nature 623, 676–677.
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3. Harrington, L.B., Burstein, D., Chen, J.S., Paez-Espino, D., Ma, E., Witte, I.P., Cofsky, J.C., Kyrpides, N.C., Banfield, J.F., and Doudna, J.A. (2018). Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science 362, 839-842.
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6. Xiang, G., Li, Y., Sun, J., Huo, Y., Cao, S., Cao, Y., Guo, Y., Yang, L., Cai, Y., Zhang, Y.E., et al. (2023). Evolutionary mining and functional characterization of TnpB nucleases identify efficient miniature genome editors. Nat. Biotechnol. 42, 745-757.
7. Saito, M., Xu, P., Faure, G., Maguire, S., Kannan, S., Altae-Tran, H., Vo, S., Desimone, A., Macrae, R.K., and Zhang, F. (2023). Fanzor is a eukaryotic programmable RNA-guided endonuclease. Nature 620, 660-668.
8. Jiang, K., Lim, J., Sgrizzi, S., Trinh, M., Kayabolen, A., Yutin, N., Bao, W., Kato, K., Koonin, E.V., Gootenberg, J.S., et al. (2023). Programmable RNA-guided DNA endonucleases are widespread in eukaryotes and their viruses. Sci. Adv. 9, eadk0171.
9. Altae-Tran, H., Kannan, S., Demircioglu, F.E., Oshiro, R., Nety, S.P., McKay, L.J., Dlakić, M., Inskeep, W.P., Makarova, K.S., Macrae, R.K., et al. (2021). The widespread IS200/605 transposon family encodes diverse programmable RNA-guided endonucleases. Science 374, 57-65.
10. Xu, X., Chemparathy, A., Zeng, L., Kempton, H.R., Shang, S., Nakamura, M., and Qi, L.S. (2021). Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing. Mol. Cell 81, 4333-4345.e4334.
11. McGaw, C., Garrity, A.J., Munoz, G.Z., Haswell, J.R., Sengupta, S., Keston-Smith, E., Hunnewell, P., Ornstein, A., Bose, M., Wessells, Q., et al. (2022). Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing. Nat. Commun. 13, 2833.https://doi.org/10.1016/j.molcel.2024.07.007

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