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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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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

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

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