Optimizing AAV Gene Delivery: Hitting the Target Safely and Effectively

The great hope for gene therapy is that one day it will be out of the clinic and into mainstream therapeutics. Bearing enormous potential to treat or cure disease, such technology can replace a defective gene with a healthy one, modulate a disease-causing one, and even introduce a gene as a form of therapy. Successes are building including recent FDA approvals for gene therapy products that treat cancer, hemophilia, and sickle cell diseases. Yet many more applications await.

One of the most actively investigated gene transfer vehicles and emerging platforms is that of adeno-associated virus (AAV). Simply put, AAV consists of a protein shell (capsid) encasing a small single-stranded DNA genome. For recombinant versions (rAAV), scientists replace the viral genome with the desired therapeutic genetic cargo. Key attributes of rAAV include its established safety profile, broad tissue tropism, and versatile manufacturing capabilities.¹

Indeed, last November the FDA approved PTC Therapeutics’ Kebilidi, the first U.S. gene therapy to be administered directly into the brain.² The AAV-based therapeutic treats a rare fatal genetic disorder characterized by a deficiency of aromatic L-amino aciydecarboxylase (AADC).

Other advances coming from clinical trials include remarkable vision improvement for rare blinding eye disorders such as Leber congenital amaurosis 1 (LCA1).³

Despite these steady successes, numerous challenges remain such as AAV’s limited cargo capacity (~ 4.7 kb), tissue specificity, and complex manufacturing. GEN spoke with experts in this field for their input as to how they are working to overcome issues to optimize AAV gene delivery.

For example, to address targeting challenges, companies are refining and optimizing capsid design via sophisticated screening platforms or via artificial intelligence (AI). Some modular platforms utilize similar capsids and manufacturing platforms yet primarily alter genetic cargo in order to treat more than one disease target. Others modify capsids to penetrate specific tissues such as the blood-brain barrier (BBB) for treating central nervous system (CNS) diseases. One piece of advice offered by a veteran scientist is to optimize early on every process involved in AAV-development technologies.

Targeting bottlenecks

In business, “location, location, location” can determine success or failure. The same could be said for AAV gene therapy. “The old adage goes that gene therapy is all about delivery, and if you are able to get your therapy to the right tissue or cell type, they will likely show a therapeutic benefit,” says Brian Kevany, PhD, CSO and CTO of Abeona Therapeutics.

According to Kevany, “Many therapies today lack a high degree of specificity to home in on a specific location or, alternatively, are attracted to off target tissues leading to unintended consequences. Further, traditional approaches of AAV capsid shuffling and rational design have not resulted in the predicted advances despite the recent involvement of AI/machine learning (ML). Thus, effective targeting remains a bottleneck to the development of AAV-based therapies.”

To overcome this targeting bottleneck companies are increasingly using higher AAV doses to reach a therapeutic benefit. These higher doses can result in potentially harmful immune responses. Kevany dismays, “Recent examples of therapies in the clinic using these high doses have resulted in vision loss, or even death.”

The company is addressing delivery problems using a multifaceted approach involving a novel capsid and a unique route of administration that only requires a lower dose. As Kevany explains, “Our proprietary AAV204 capsid has been shown to efficiently transduce cells of the retina upon injection directly into the vitreous cavity of the eye. For our lead preclinical program, for X-linked retinoschisis (XLRS), we are taking advantage of this capability and administering it using a para-retinal injection. This method is less invasive than a traditional subretinal injection and also positions the therapy closer to its intended target and at a lower dose than what is used in a standard intravitreal injection.”

The XLRS therapy is intended to treat patients with mutations in the RS1 protein that result in deleterious splitting of the layers of the retina, ultimately leading to vision loss. The therapeutic payload of this drug is a corrected version of the RS1 protein driven by a photoreceptor-specific promoter.

Abeona’s development pipeline also includes AAV-based therapies targeting Stargardt Disease and Autosomal Dominant Optic Atrophy.

Crossing the BBB

“The BBB prevents the uptake of many investigational therapies, including in gene therapy,” remarks Todd Carter, PhD, CSO, Voyager Therapeutics. According to Carter, the company is seeking to overcome these challenges through their TRACER™ (Tropism Redirection of AAV by Cell-type-specific Expression of RNA) capsid discovery platform. He reports, “This is an RNA-based screening platform that has allowed us to create multiple families of novel, IV-delivered AAV capsids demonstrating robust penetration of the BBB. Our capsids have been shown to transduce a broad range of CNS regions and cell types, with decreased transduction of the liver. Further, we’ve seen cross-species CNS tropism (including rodents and multiple non-human primate species) resulting in widespread payload expression across the CNS at relatively low doses.”

Following the robust performance of the TRACER platform, the company has selected five gene therapy development candidates. Carter discloses, “We feel that 2025 is therefore going to be a very important year for us. By mid-2025, we plan to submit an IND for our TRACER-derived capsid with vectorized anti-SOD1 siRNA for the treatment of amyotrophic lateral sclerosis (ALS). Also in 2025, we expect IND filings by our partner Neurocrine in one program addressing Friedreich’s ataxia and a second program for Parkinson’s disease and other GBA1-mediated disease.” Mutations in GBA1, the gene encoding glucocerebrosidase, are among the most common genetic risk factors for development of Parkinson’s and other related diseases.

Looking to the future, Carter says the company is aiming for an even bigger impact. “We’re excited about the potential of leveraging the receptors we’ve identified through our AAV capsids to shuttle other macromolecules across the BBB in a manner that is non-viral. We expect that each receptor could have its own profile in pharmacokinetics and safety to provide opportunities in different diseases and indications. We’re still in the preclinical stages, but ultimately, we aim to use these different approaches to solve the fundamental problem of CNS delivery that the field has struggled with, and to expand into other modalities of neurogenetic medicine broadening our impact.”

 AI-assisted AAV design

Recent advances in generative AI are beginning to revolutionize AAV capsid design. Eric Kelsic, PhD, CEO and cofounder, Dyno Therapeutics weighs in, “Optimizing AAV capsids for efficiency, specificity, and reduced immunogenicity is an immensely complex task in itself. Dyno was the first company to recognize that ML, when supported by the right data, is uniquely suited to address this problem.”

According to Kelsic, new AI-assisted models can be trained to generate AAV capsids with more highly optimized properties compared to other capsid design methods. “The key advantage is that they leverage vast datasets with diverse input sources ranging from experimental studies to protein databases, to access and navigate areas of the sequence space that cannot be explored by traditional approaches.”

Kelsic explains that the company’s ML models produce and test billions of candidate sequences in silico, filtering out nonviable sequences and advancing the most promising candidates. “Our LEAP^SM (Low-shot Efficient Accelerated Performance) technology allows engineers to bypass entire rounds of experimental de-risking, replacing years of experiments with a few hours of computation. This year we reported that 9 out of 19 novel capsids designed using LEAP outperformed any of our previously measured capsid sequences with respect to brain transduction and liver detargeting, and that a small number of capsids designed in silico using LEAP could match the performance of an experimental capsid design round testing millions of capsids using traditional directed evolution methods.”

The pioneering technology is “validated and already demonstrating value for partners,” Kelsic reports. He continues, “We recently showcased the Dyno eCap^TM 1 capsid, which achieved an 80-fold improvement in gene delivery efficiency to the eye, and the Dyno bCap^TM 1 capsid, which demonstrated a 100-fold improvement in delivery to the brain. Both capsids are available for licensing to partners who have payloads ready to go.”

Dyno’s partnership-centric model aims to maximize the reach and overall patient impact of their technology. Kelsic summarizes, “By working collaboratively with industry leaders, we ensure that our innovations benefit the entire gene therapy ecosystem, rather than being confined to a select few therapeutic pipelines.”

Modular platform

Following approval of their groundbreaking AAV-based gene therapy for hemophilia B (HEMGENIX^@), uniQure continues to optimize their technologies and address challenges such as how to provide a more focused delivery, increase cellular transduction, and overcome pre-existing neutralizing antibodies.

The company is targeting liver and CNS disorders employing a array of tools. Their “Smart AAV” technology provides novel capsids for CNS delivery that utilize antibodies and peptides for specific targeting and for crossing the BBB. Cargo-specific technologies include miQURE, a single gene silencing platform, LinQURE that provides multiple gene-silencing microRNAs in a single AAV, and GoQURE that simultaneously knocks down a diseased gene and replaces it with a healthy version.

The company has several candidates in clinical trials. Their Huntington’s disease-targeting AMT-130 (an AAV-encoding miRNA that non-selectively lowers huntingtin protein) is undergoing PhaseI/II trials. According to Walid Abi-Saab, MD, chief medical officer, the company has reached agreement with the FDA on core components of an Accelerated Approval pathway for AMT-130 and has initiated Biologics License Application (BLA) activities. A gene therapy candidate for treating temporal lobe epilepsy, AMT-260, is undergoing Phase I/IIa testing. AMT-260 provides local delivery of miRNA silencing technology against the GRIK2 gene that encodes epilepsy-triggering receptors. Another therapy, AMT-162, in PhaseI/IIa trials, targets amyotrophic lateral sclerosis (ALS) resulting from SOD1 mutations. The miRNA-containing AAV vector serves to knock-down mutant SOD1 as a one-time administration.

Prioritizing process optimization

“Every gene therapy company should optimize and develop every technology in their arsenal of viral vectors,” advises Alexandria “Zandy” Forbes, PhD, president and CEO MeiraGTx. She continues, “We started out about nine years ago focusing on diseases which we could address using local delivery of small doses of optimized vectors to achieve a clinical impact, rather than focusing only on inherited genetic diseases.”

Since manufacturing AAV’s can be challenging, Forbes says they decided early on to tackle each facet of the process. She reports, “We started one of the first GMP facilities in the U.K. and wanted it to be a premier manufacturing company that is both flexible and scalable. We created a single GMP-ready platform process based on greater than 20 vectors with datasets from hundreds to thousands of conditions. This allows us to provide the highest yield in the industry and bring down the cost of goods.”

Another strategy the company used to enhance efficiency was to develop and validate their own internal quality control (QC) assays. Forbes notes, “We found during the clinical development of our early programs that using outside vendors for QC is very rate-limiting, causing delays of weeks to months. Therefore, we built our own QC facility which now has a commercial license, as well as aligning with the regulatory agencies globally on a path to commercial manufacturing approval. Overall, this strategy provides a robust manufacturing platform for clinical material and shaves years off the clinical development timeline for these products. We currently have four late-stage clinical programs with potential BLA filings expected in 2025, 2026, and 2027.”

According to Forbes, one of the most exciting recent developments is the company’s Riboswitch technology that allows the precise control of gene expression using an oral medication. She concludes, “Thus, taking a simple pill could allow us to precisely control the amount of any gene product produced in the body, be it antibody or peptide. This technology allows the delivery of naturally occurring fast-acting agonists such as those involved in the control of metabolism, which opens an entirely new approach to the selection of therapeutic biologic targets compared to the industry-wide history of focusing on inhibitors.”

References

1. Wang, J-H et al. Adeno-associated virus as a delivery vector for gene therapy of human diseases. Signal Transduction and Targeted Therapy (2024) 9:78.
2. www.fda.gov/news-events/press-announcements/fda-approves-first-gene-therapy-treatment-aromatic-l-amino-acid-decarboxylase-deficiency
3. www.genengnews.com/topics/drug-discovery/aav-gene-therapy-for-lca1-improves-vision-dramatically/