Gene and cell therapies continue to advance, providing solutions to rare monogenic diseases and common conditions. Cellular therapies, best represented by CAR-T cells, have drastically changed the outcomes in hematological malignancies and spurred more effective solutions for solid tumors. However, one significant obstacle to maximizing the potential of gene therapies and editing technologies is the capacity to target specific organs, tissues, and cells in vivo. Here we discuss three approaches presented at ASGCT 2023, which provide new options for targeted in vivo cargo delivery of genes, base editors, and Cas9 RNPs.
Pushing the boundaries of what systemic gene therapy can accomplish, scientists at Regeneron are developing new strategies for targeted gene delivery. By leveraging Regeneron’s strengths in monoclonal antibody development, they aim to bridge the viral tissue-targeting gap. In this new approach, AAV particles are covalently bound to monoclonal antibodies that target tissue-specific surface proteins.
Leah Sabin, Director, Viral Vector Technologies at Regeneron Pharmaceuticals
How do they achieve this? Regeneron has adopted the SpyTag-SpyCatcher technology for protein ligation developed by the Howarth lab and first reported in 2012 (Zakeri et al. 2012). By engineering AAV capsids to express SpyTag and tagging antibodies with SpyCatcher, Regeneron has developed a platform for the covalent modification of AAV particles.
SpyTag/SpyCatcher system. The SpyTag (13 aa) and SpyCatcher (15 kDa)technology enable spontaneous, rapid, and irreversible conjugation of proteins. Retrieved without modifications from: https://en.wikipedia.org/wiki/SpyCatcher (https://creativecommons.org/licenses/by-sa/4.0/) Right: Created with BioRender.com
Additionally, to further ensure safety and improve efficacy, AAV capsids themselves have been evolved to reduce liver targeting. Essentially, this new modular platform provides expanded opportunities to fine-tune two critical aspects limiting the success of current viral delivery strategies. So far, AAV candidates re-directed to the skeletal muscle dihydropyridine receptor have demonstrated robust skeletal muscle targeting while successfully de-targeting cardiac muscle and the liver. Because antibody-conjugated AAVs are tested in vivo in humanized mouse models, Regeneron’s approach has the potential for faster translatability.
Base editors make precise single nucleotide changes (e.g., A.T to G.C), enabling genomic editing to correct mutations or silence genes linked to disease states. The precision of base editors and their mechanism of action, which circumvents double-strand breaks, provide the opportunity to correct critical mutations in many monogenic diseases. However, as with all CRISPR tools, in vivo delivery represents the ultimate challenge (Reshetnikov et al. 2022).
Katarina Kulhankova, Research Scientist, University of Iowa
A new approach developed by scientists at the University of Iowa leverages the unique properties of shuttle peptides to deliver base editor RNPs in vivo directly to the lungs as a potential gene-editing therapy for Cystic fibrosis (CF).
Shuttle peptides for the delivery of Cas9 RNPs to airway epithelia. Shuttle peptides consist of two critical domains: a cationic domain that promotes cell internalization (Cell Penetrating Peptide-CPP) and a hydrophobic domain, or endosomolytic peptide (ELD) sequence, to safeguard the editing cargo from endosomal sequestration. Retrieved (Figure 1, panel b) from Kulhankova et al. 2023) https://creativecommons.org/licenses/by/4.0/
Why target the lungs? Although the mutated gene in CF, CFTR, is expressed in different tissues, its malfunction in the lungs is central to the pathological changes resulting in cardiac failure and mortality. Additionally, the accessibility of the airway route makes the lungs an attractive target for delivering gene-editing cargo (Kotzamanis et al. 2013). Still, the airway epithelia have presented a significant challenge for gene therapy in CF, improving CFTR function insufficiently and without clinical benefits.
Why shuttle peptides? The McCray lab had already implemented this approach, successfully delivering CRISPR RNPs to mouse lungs in vivo. They previously observed that shuttle peptides offer advantages over other delivery vehicles, such as fast cargo delivery and rapid peptide turnover. Shuttle peptide-delivered Cas9 and Cas12a RNPs in vivo to the mouse lungs achieved similar editing efficiencies in the airways of ~12% (Krishnamurthy et al. 2019). Now, in their most recent work, the group has taken a significant step forward by successfully shuttling base editor RNPs to rhesus monkey airway epithelial cells in vivo to effect gene editing (Kulhankova et al. 2023).
In continuing to advance opportunities for in vivo CRISPR genome editing, the Doudna lab is taking a new approach that leverages the strengths of viral particles as cargo delivery vehicles. T-cell engineering has been transformative in producing innovative and effective cancer therapies, particularly for hematological malignancies. Yet, the workflow for T cell engineering is complex and challenging as it involves the harvesting, editing, and re-infusion of T cells. In fact, harvesting T cells from some patients is not at all feasible due to their T cell health or low counts (Xu et al. 2020).
Jennifer Hamilton, Postdoctoral Fellow, University of California, Berkeley
Recognizing this significant limitation, one goal of their work is to develop strategies to simplify and increase the success of T-cell engineering processes by doing all the editing in vivo. How are they doing this? By engineering lentivirus to ① package Cas9 RNPs and CAR transgenes, ② deliver their cargo precisely to T cells, and ③ activate T cells (Hamilton et al. 2023).
Cas9 RNP delivery vehicles. Lentivirus engineering to target T cells in vivo, providing all needed components for knockout of the endogenous T cell receptor and CAR transgene delivery. Created with BioRender.com
First, to trick lentivirus into packaging RNP complexes, Cas9 has been fused to the viral structural protein Gal, which is typically involved in packaging the viral RNA. Next, the surface of these Cas9 delivery vehicles was engineered to express molecules for specific T-cell targeting and endosomal fusion. Additionally, to induce activation and expansion of T cells, these particles also display scFvs such as anti-CD3 and anti-CD28. Lastly, with packaged CAR transgenes, this new system elegantly integrates all the required components to manufacture a T cell therapy in vivo, which has been successfully achieved in mice.
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