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EPL: An Enhancer Polymer for Improved AAV Gene Therapy Targeting and Safety

Introduction to AAV Gene Therapy Challenges

  • AAV Capsid Engineering: Capsids are continuously engineered to improve properties such as targeting, immunogenicity, and safety.

  • FDA-Approved AAVs: Several AAV serotypes are FDA-approved and utilized in clinical-grade therapies. Examples include AAV9 and AAVRH74.

  • Challenges of Systemic AAV Gene Delivery:

    • Safety Concerns: High doses of systemic AAV gene delivery are notoriously unsafe.

    • Liver Tropism & Immune Response: Irrespective of the AAV serotype, intravenous (IV) infusion typically results in preferential delivery to the liver.

      • This preferential liver delivery triggers an immense immune response.

      • Patients receiving such therapies frequently undergo immunosuppression to mitigate this response.

    • Adverse Events: Adverse events, sometimes leading to patient deaths, have been reported in clinical trials.

      • A recent example includes the FDA suspending an AAV gene therapy trial for Duchenne muscular dystrophy due to patient deaths.

  • Research Goals: The primary objectives are to enhance gene therapy targeting, reduce immunogenicity, and boost safety.

  • Proposed Solution: Enhancer Polymer (EPL): An engineered polymeric nanoparticle designed to be intravenously injected before AAV administration.

    • EPL aims to improve AAV targeting and diminish immunogenicity.

Enhancer Polymer (EPL): Solution and Mechanism

  • EPL's Role in AAV Delivery:

    • Without EPL: AAV is rapidly sequestered, resulting in minimal delivery to the intended target organ.

    • With EPL: AAV delivery to the organ of interest is significantly improved.

  • Cardiac Gene Therapy Example:

    • Traditional AAV: When injected intravenously, most of the drug accumulates in the liver.

    • With EPL: Separate injection of EPL enables detargeting from the liver and enhanced targeting of the heart.

    • This strategy is referred to as a "bait and switch" approach, where EPL acts as a decoy for the immune system, which would otherwise clear AAV.

  • Nature of EPL:

    • Synthetic Polymer: EPL is a synthetic polymer developed in the lab through high-throughput screening.

    • Composition: It is a poly(lactic-co-glycolic acid) (PLGA) based polymer, utilizing two monomers.

    • Biodegradability and Safety: EPL integrates easily in vivo, degrading into safe and non-immunogenic lactic and glycolic acids. It is a well-established polymer in the industry.

  • Administration Timeline:

    • EPL is injected at t=0 hours.

    • AAV gene therapy is administered 15 to 30 minutes later.

    • Therapeutic delivery and most transduction occur within 1 to 2 hours, owing to the high efficiency of AAVs/viruses in transducing cells.

  • Suitability and Excretion:

    • Ideal for single-shot gene therapies.

    • Safe and eliminated primarily via hepatobiliary excretion.

    • Extensively tested for cardiac delivery, with limited data for other target organs.

EPL Application and Serotype-Dependent Effects

  • AAVRH74 (Cardio-Muscle Tropic) Data:

    • Experimental Setup: Administered AAVRH74 (carrying a GFP transgene) at a low dose (5 \times 10^{12}) with or without EPL in wild-type mice. GFP fluorescence in heart and liver was observed.

    • Heart Transduction: EPL drastically enhanced GFP transduction in the heart. Vehicle-injected AAV failed to achieve significant transduction at this low dose.

    • Liver Detargeting: EPL facilitated liver detargeting, leading to a significant increase in heart targeting.

    • Quantitative Analysis:

      • Western Blotting (GFP protein level): Achieved up to an order of magnitude higher heart-to-liver ratio with EPL compared to vehicle only.

      • PCR (viral DNA): Demonstrated nearly a magnitude difference for liver detargeting, with EPL significantly increasing heart targeting.

  • Brain Detargeting: EPL injection also resulted in brain detargeting by AAV, which could be beneficial for safety.

  • AAV1 Data:

    • Inherent Challenge: AAV1 is known for poor in vivo performance, despite good in vitro efficacy, due to rapid liver deposition (half-life of approximately 1 minute).

    • EPL Effect: EPL significantly improved heart delivery of AAV1 and detargeted the liver. PCR analysis of viral DNA showed a similar trend in heart-to-liver ratio improvement.

    • In some vehicle-injected mice, AAV1 DNA transcripts were undetectable, highlighting EPL's impact.

  • AAV9 Data:

    • Inherent Tropism: AAV9 is often considered heart-tropic, but literature indicates it is more effective at infecting the liver and is generally more infectious than other serotypes.

    • EPL Effect (Luciferase Transgene, Low Dose): With EPL, there was very high deposition of AAV9 into the liver and almost no transfection efficiency with regular AAV (observed at a short time point, 1 week).

    • EPL detargeted the heart with AAV9 and increased AAV9's liver specificity.

  • Conclusion on Serotype Dependence:

    • EPL technology is serotype-dependent, enhancing the inherent tropism of the AAV.

    • If a serotype is heart-tropic (e.g., AAVRH74, AAV1), EPL improves heart targeting (and detargets the liver).

    • If a serotype is liver-tropic (e.g., AAV9), EPL improves liver targeting (and detargets the heart).

Deep Dive into EPL's Mechanism of Action

  • Complexity: The mechanism of action is complex and involves multiple compartments (heart, liver, systemic circulation). Ongoing research is exploring various aspects.

  • EPL Biodistribution:

    • When labeled with a Sci-Fi fluorophore, EPL (after IV administration) is primarily deposited in the liver; none goes to the heart.

    • Sci-Fi alone distributes uniformly across organs.

  • Cellular Uptake of EPL:

    • Hepatocytes: Do not take up much EPL in vitro.

    • Macrophages: Rapidly take up EPL (consistent with its nanoparticle nature).

    • Flow Cytometry (Liver Cells): EPL is predominantly taken up by cytokine-releasing Kupffer cells, more so than T cells, neutrophils, hepatocytes, or liver sinusoidal endothelial cells.

  • Route of Administration: Although most studies use IV delivery, intraperitoneal (IP) administration was tested, showing different distribution patterns, reinforcing the important role of Kupffer cells.

  • Modulation of AAV Cell Entry:

    • Given the similar trends in viral DNA and protein expression, it is highly probable that EPL modulates the initial step of AAV cell entry: receptor binding.

    • AAV cell entry pathway: Receptor binding ( \rightarrow ) Endosomal encapsulation ( \rightarrow ) Endosomal escape ( \rightarrow ) Nuclear translocation ( \rightarrow ) Viral DNA release ( \rightarrow ) Episomal DNA assembly ( \rightarrow ) Transcription/Translation.

  • AAV Receptors and Glycosylation:

    • Diverse Receptors: AAV receptors are numerous, serotype-dependent, and can involve co-receptors.

    • Common Feature: Glycosylation: A prevalent characteristic among AAV receptors is glycosylation, specifically modification by sialic acid.

  • EPL's Impact on Whole-Body Metabolism:

    • Fluorodeoxyglucose (FDG) PET Imaging: Used to determine real-time glucose uptake in organs.

    • Results: EPL injection (followed by FDG) led to a highly prominent glucose uptake in the heart, while decreasing glucose uptake by the liver.

    • Brain glucose distribution remained unchanged, suggesting a potential safety advantage.

  • Glucose Metabolism and Glycosylation in the Heart:

    • Glucose-to-Mannose Conversion: The increased glucose metabolism in the heart with EPL is linked to the transformation of glucose into mannose.

    • Western Blotting (Heart Lysates): EPL-injected animals showed very high mannose expression in heart lysates, detectable with specific antibodies.

    • SLC Transporter: The SLC transporter, crucial for mannose transport in the heart, is also regulated by EPL.

    • Tissue Glycosylation: Using lectins (e.g., MAL2), highly glycosylated tissue was detected in the heart after EPL administration.

    • Conclusion: EPL increases cardiac glucose uptake, which drives mannose conversion and subsequent tissue glycosylation. These glycosylations serve as critical AAV receptors.

  • How EPL Increases Cardiac Glucose Uptake (Calcium Signaling):

    • In Vitro Macrophage Experiment: Bone marrow-derived macrophages were cultured, fed EPL, and their supernatant collected.

    • Cardiomyocyte Assay: Primary mouse cardiomyocytes were treated with this supernatant, and sarcomere shortening and calcium transients were measured.

    • Results: EPL supernatant did not affect sarcomere shortening but drastically altered calcium transients in cardiomyocytes.

    • Comparison: EPL itself and its degradation byproduct, lactate, did not yield the same results.

    • Mechanism Link: Calcium activation triggers the enzyme PDP, which dephosphorylates pyruvate dehydrogenase (PDH).

      • This dephosphorylation switches cardiomyocyte metabolism from fatty acids (preferred) to glycolytic metabolism (less common).

      • EPL's prominent effect on calcium uptake in cardiomyocytes leads to increased phosphorylated PDH, indicative of heightened glycolytic metabolism.

Pharmacokinetics Improvement and Broad Utility

  • Second Mechanism: AAV Pharmacokinetics (PK): Prolonging AAV circulation time can enhance tissue infection efficacy.

  • AAV9 PK Study:

    • Experimental Setup: EPL was injected, followed by AAV9. Blood sampling over 24 hours allowed for viral DNA detection in circulation.

    • Results: EPL dramatically improved the pharmacokinetic profile of AAV9.

    • Half-Life Increase: The half-life of AAV9 was approximately 4-fold longer with EPL administration.

  • Hypothesized PK Mechanism: EPL is thought to improve PK by blocking reticuloendothelial system (RES) cells, such as Kupffer cells and macrophages, either in systemic circulation or the liver. This overwhelms the RES, allowing AAV to circulate longer (currently under investigation).

  • Broader Applications Beyond AAV:

    • Polymeric Nanoparticles: EPL improves the PK profile of other polymeric nanoparticles.

    • Lipid mRNA-Carrying Nanoparticles (LNPs):

      • Pancreatic Cancer Model: Injected non-modified LNPs (containing mRNA) directly into the pancreas with or without EPL.

      • Results: With EPL, clear on-tumor delivery and liver detargeting of LNPs were observed.

      • This demonstrates that EPL can improve targeting to multiple tissues/organs beyond heart/liver therapies, simply by changing the pharmacokinetics of the therapeutic agent.

    • Cell Therapies: EPL has been tested with mesenchymal stem cells and CAR T cells, among other modalities.

Safety Profile and Clinical Translation

  • Importance of Safety: Crucial for translating therapies to humans.

  • Non-Human Primate (NHP) Studies:

    • Collaboration: Performed in collaboration with Wake Forest University, along with mouse data.

    • Rationale: NHPs offer an immune system and other features that closely resemble humans, unlike mice.

    • Protocol: Three vervet monkeys were injected with EPL. Serum samples were collected before and after injection and analyzed for immune response, liver toxicity, and other metrics.

  • NHP Safety Results:

    • Blood Biochemistry: Absolutely no changes were observed in common blood biochemistry parameters (e.g., albumin, creatinine, ALT, AST, cholesterol, triglycerides, urea) at 2 time points.

    • Chemokine/Cytokines (Immune Response): No significant immune response was detected. Only VGF was slightly upregulated after 7 days in EPL-treated mice (a specific finding).

    • Complement Activation: This is a critical factor in AAV therapies, as most induce high complement activation.

      • C3a levels remained unchanged after EPL administration.

      • C5a levels were decreased 7 days after administration, which is counter-intuitive and highly significant, as immunogenic therapies typically increase complement activation.

Broader Applications and Future Directions

  • Cardiac Diseases: Potential applications include various cardiac conditions, such as Friedrich's ataxia (though serotype procurement was an issue in initial attempts).

  • Liver Diseases: Applicable to numerous rare liver diseases.

  • Universal Approach: EPL technology can be integrated with various modalities, including nanoparticles and cell therapies (e.g., CAR T cells, NK cells).

  • Beyond Cardiac and Liver: Potential applications extend to cancer, lung disease, and other areas.

Q&A Insights

  • Q: Osmolarity/Viscosity Changes?

    • A: While theoretically possible, it's unlikely with the low EPL doses (30\, \text{mg/kg}) and volumes (100\,\mu\text{L/mouse}) used, as they are comparable to monoclonal antibodies or small molecules and unlikely to significantly alter blood osmolarity.

  • Q: AAV9's Reverse Distribution Mechanism?

    • A: EPL enhances the inherent tropism of the AAV serotype. AAV9 is a liver-tropic serotype. By increasing AAV's pharmacokinetic half-life, EPL prolongs its circulation, leading to extended contact with the liver and thus increased liver uptake, overriding any heart-tropic effects observed in some studies.

  • Q: Identifying Active Component in Macrophage Supernatant?

    • A: Proteomics is underway to identify the component responsible for the observed effects. If successful, this isolated component could potentially replace EPL.

  • Q: Brain Delivery for Neurological Diseases?

    • A: Yes, for diseases with neurological implications (e.g., Friedrich's ataxia), achieving brain delivery is a goal. Some newer engineered AAV serotypes are known to cross the blood-brain barrier. Testing these with EPL could enable dual delivery to the brain and other target organs.

  • Q: Why are most AAV Serotypes Liver-Tropic?

    • A: The liver is a large organ, and AAVs are viruses. The immune system generates antibodies that sequester AAV, forming immune complexes that are efficiently cleared by the liver. This is a primary mechanism for rapid viral clearance.

  • Q: Safety of Nanoparticles?

    • A: Safety depends on the specific nanoparticle and dose. Increased complexity in nanoparticle engineering can raise safety concerns. While some nanoparticles (e.g., LNPs in vaccines) are generally considered safe, they can become toxic at drastically high doses (e.g., 1000\times), as the constituent lipids themselves can be toxic at excessive concentrations.

  • Q: Direct Influence of EPL on Cardiac Function?

    • A: EPL itself does not biodistribute to the heart. However, paracrine effects are observed. Serum from EPL-injected mice or monkeys (containing a secretome released by the liver or other organs in response to EPL) has been shown in vitro to increase AAV transduction efficiency in cardiac cells, indicating an indirect influence via a circulating entity.

  • Q: Exosome-Mediated Trafficking from Liver to Heart?

    • A: This is a key area of investigation. It's plausible that a secondary messenger, such as an exosome, a specific protein, or a cytokine, is produced by the liver in response to EPL, released into the serum, and then influences receptor expression in the heart, leading to increased AAV transduction.

  • Q: How was EPL discovered?

    • A: The discovery was accidental. A biological effect was observed during nanoparticle screening, recognized as potentially useful, and then followed up with high-throughput screening of different polymers to arrive at the current EPL formulation.

  • Q: Testing with Engineered AAV Serotypes?

    • A: Yes, collaborations are in place with researchers who possess engineered AAV serotypes (some of which completely detarget the liver). Future plans include testing EPL with these advanced serotypes to further enhance heart or muscle transduction, potentially at even lower AAV doses, which would be highly significant.