Reprogramming Astrocytes to iNeurons Using the iPhAGE System

Overview

Neurodegenerative diseases, characterized by the irreversible loss and dysfunction of neurons, pose a significant and growing global health concern. The prevalence of these age-related neurological disorders is steadily increasing, with an estimated 1.5 billion people over 65 by 2050. These conditions are the leading cause of disability worldwide, leading to severe impairments in motor, sensory, and cognitive functions. Currently available treatments primarily manage symptoms with limited success in halting or reversing disease progression, with no truly effective and curative therapies available.

Gene therapy offers a promising avenue for treating neurodegenerative disorders, particularly within the central nervous system (CNS), however, faces significant challenges including:

• Crossing the Blood-Brain Barrier (BBB): The BBB is a highly selective endothelial cell border encompassing the neural vasculature that protects the brain from toxins and maintains a stable environment within the CNS. However, this protective function poses a significant challenge for gene therapy as it often prevents the delivery of therapeutic vectors to affected neural tissues.

• Mitigating Immune Responses: Managing potential immune responses to the vectors delivering genetic material and the genetic material itself is crucial for long-term efficacy and safety.

• Precise Cell-Targeting: Ensuring precise targeting of affected cells while avoiding unintended off-target effects is paramount for successful gene therapy.

Team iNeuron: Exploring the Potential of Neuronal Reprogramming

Team iNeuron is at the forefront of exploring neuronal reprogramming as a groundbreaking treatment strategy for incurable neurological disorders. As a part of this international team, our core aim is to directly convert existing brain cells into healthy new neurons, termed “iNeurons.” This revolutionary method holds immense promise for treating a wide range of neurological conditions stemming from neuronal loss or dysfunction.

The iNeuron Project is focusing its development efforts on three critical disease models, including Alzheimer’s Disease, Chronic Stroke, and Intractable Epilepsies. Team iNeuron is structured into three specialized pillars:

• Pillar 1: Team Cargo - focused on the gene therapy cargo that will optimize neuronal reprogramming

• Pillar 2: Team Delivery - focused on the development of astrocyte-specific delivery systems

• Pillar 3: Team Translation - focused on assessing outcome measures

As a part of Pillar 2: Team Delivery, our lab is exploring the potential applications of the iPhAGE system for delivery of neuronal transcription factors to astrocytes.

Team iNeuron actively engages with patients and individuals with lived experience through the Advisory Board for Lived Experience of Stroke, Epilepsy and Dementia (ABLE), which plays a crucial role in the iNeuron project by providing invaluable patient and caregiver perspectives. Their input ensures that our research on neuronal reprogramming is truly patient-centered and addresses the most meaningful outcomes for those living with these neurological disorders.

Using the iPhAGE System to Enable Targeted Neuronal Reprogramming

Specifically, the M13 iPhAGE System offers several key advantages for neuronal applications:

• CNS-Targeted Delivery: The M13 bacteriophage, a single-stranded DNA phage, possesses a well-documented ability to cross the blood-brain barrier. This inherent trait is critical for targeted phage delivery to the CNS, enabling us to overcome a major hurdle in gene therapy for neuronal disorders.

• Targeted Delivery: Modifiable targeted ligand display confers the ability to target and penetrate specific cell types, allowing for localized vector delivery.

• Enhanced Safety & Purity: Our process entirely removes the bacterial backbone from the ssDNA vector, resulting in phagemids that are free from prokaryotic genes that may induce immune responses. This significantly reduces immunogenicity and results a highly pure final product, addressing long-standing limitations of traditional plasmid and phage-based vector applications for CNS disorders.

Reactive astrocytes (or “activated astrocytes”) develop as a protective and adaptive mechanism in response to pathological stimuli within the CNS, including ischemia, injury, disease, or inflammation. They are distinct from homeostatic astrocytes due to their hypertrophic phenotype, increased ETBR expression, and functional changes. This reactive state makes them ideal targets for gene therapy in neurodegenerative disorders for several key reasons:

• Abundant at Disease Sites: Reactive astrocytes are highly prevalent in areas affected by disease or injury, ensuring that therapeutic delivery can be precisely localized where needed most.

• Leveraging Upregulated Genes: Their reactive state involves the upregulation of specific genes, including endothelin B receptors (ETBR), which can be leveraged for targeted gene delivery, ensuring therapeutic agents primarily address diseased regions while sparing healthy, non-reactive astrocytes.

• Localized Intervention & Reprogramming: By targeting reactive astrocytes, we can achieve localized intervention within the pathological environment. This offers the potential to convert them into iNeurons, without broadly impacting the entire astrocyte population, which is necessary for typical neural functions in healthy brain regions.

Current Work & Progress

Our overall goal is to demonstrate that intravenous administration of engineered miniphagemid ssDNA vectors are able to effectively cross the blood brain barrier and specifically deliver neuronal reprogramming transcription factors to astrocytes, resulting in the differentiation of reactive astrocytes to iNeurons.

Alterations to the iPhAGE System for CNS Applications:

To optimize the iPhAGE system for this specific application, several modifications were made:

• The precursor plasmid provides the gene cassette (including the gene of interest) that is packaged within the miniphagemid vector.

  • Modified to encode the Neurogenic Differentiation Factor 1 (NeuroD1) gene, a transcription factor that promotes neuronal differentiation, and luciferase (luc) or green fluorescence protein (gfp) for imaging and tracking.

  • Modified to encode the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter to drive the expression of the gene cassette specifically in astrocytes.

• The helper plasmid provides the phage proteins necessary for miniphagemid assembly and cell specific targeting.

  • Modified for display of the ETBR-specific ligand, IRL-1620, to selectively target and bind to activated astrocytes.

Transfection Studies:

Precursor plasmids and iPhAGE miniphagemids are transfected into immortalized astrocytes (A7 rat hippocampal cells) as well as HEK293T cells and melanocytes. To model reactive astrocytes for targeted gene delivery, A7 cells are activated using lipopolysaccharide to upregulate the expression of ETBR on the astrocyte surface.

Neurons differentiated from astrocytes are detected through staining with PSA-NCAM1 antibody conjugated to a fluorophore at day 7 and day 14 post-transfection.

Future Work:

Our lab's ongoing contributions to the iNeuron project will focus on the following key areas:

• Optimizing Gene Cassettes: Investigating alternative gene cassettes, exploring various transcription factors (e.g. Sox-2) and different targeting ligands to enhance reprogramming efficiency and specificity.

• Preclinical In Vivo Studies: Conducting in vivo animal studies using diverse neurodegenerative mouse models to evaluate the therapeutic efficacy of our approach.

• Strengthening Collaborations: Continued collaborations withDr. Carol Schuurman’s Lab (Pillar 1), Dr. Cindi Morshead’s Lab (Pillar 3), as well as Dr. Giacomo Masserdotti and Dr. Magdalena Götz’s Lab (Pillar 1), among other research partners.