Novel Anellovirus-Based Vector Production Biomanufacturing Platform

Overview

The clinical translation of gene therapy is limited by challenges associated with current delivery systems. While viral vectors demonstrate efficient transduction and transgene expression, safety concerns and high production costs limit their utility. Non-viral vectors, though safer and easier to produce, typically lack sufficient transgene delivery efficiency. These limitations underscore the need for novel delivery systems that are safe, efficacious, and provide greater transgene expression control via vector redosability and titratability.

We have engineered a novel system for the production of anellovirus-based gene delivery vectors, (“anellomids”). These vectors are formed through the self-assembly of ssDNA vectors with anellovirus capsid proteins produced by E. coli. This innovative approach enables the scalable generation of circular ssDNA vectors encapsulated within anellovirus-based particles, offering a promising solution to current gene delivery limitations. This platform demonstrates significant advantages, including:

• Enabling the scalable production of anellomids through the in vitro assembly of anellovirus capsid proteins into functional particles, demonstrating the scalability and feasibility of cost-effective manufacturing for anelloviral vectors.

• Substantial improvements in gene expression, including yields of ssDNA-based anelloviral vectors that achieve a 100- to 1000-fold improvement in gene expression in mammalian cells (HEK 293T) compared to molar equivalents of conventional dsDNA and ssDNA vector delivery.

This system is a crucial step towards unlocking the full therapeutic potential of anelloviruses for gene therapy, and early development efforts are now focused on a streamlined, single-step production strategy entirely within E. coli.

The Anellomid System at a Glance:

The anellomid system is a novel, highly efficient, and cost-effective gene therapy delivery platform. It is designed to overcome critical limitations in gene therapy by providing a safe and efficacious delivery system with greater control over transgene expression through vector redosability and titratability.

Anelloviruses have a small icosahedral capsid structure composed of 60 copies of the capsid protein. These proteins form a positively charged jelly roll domain core that facilitates self-assembly around the negatively charged ssDNA genome, with spike domains extending outwards to create a crown-like structure around the 5-fold symmetry axis of the particle. Utilizing the iPhAGE System, M13-processed circular single-stranded DNA (ssDNA) vectors containing the gene of interest are expressed by E. coli and purified. Separately, TTV19 anellovirus capsid proteins production is induced in E. coli. These components are then combined and self-assemble to form the anellomid vectors

Key features:

• Scalable production: The system allows for the scalable generation of circular ssDNA vectors encapsulated within anellovirus-like particles.

• Minimizing contamination: The ssDNA production method minimizes the risk of bacterial backbone contamination, enhancing safety and purity.

• Cost-effectiveness and efficient production: Production exclusively utilizing bacterial systems significantly reduces the overall time, labor, and cost required for large-scale production.

• Broad tropism: Anelloviruses naturally transfect a wide range of human cells and tissues, offering broad applicability.

The One-Step Anellomid Production System:

We have also begun development a novel one-step in vivo system to produce anellomids entirely within E. coli. This system offers a rapid, scalable, and adaptable solution for anellomid therapeutic development and manufacturing:

• Eliminating the need for in vitro assembly processes.

• Further minimizing the risk of backbone contamination

• Streamlining production efficiency

• Further reducing the overall cost and time required for large scale production

Production Process:

1. Production of capsid proteins and gene expression: E. coli is engineered to produce anellovirus capsid proteins simultaneously with the production of a backbone-free, single-stranded DNA minivector containing only a eukaryotic expression cassette.

2. Self-assembly in vivo: The circular ssDNA vectors self-assemble with the E. coli-produced anellovirus capsid proteins in vivo, forming anellomids.

3. Purification: The assembled anellomids are then purified from the E. coli environment.

Early data indicates that the efficiency of the in vivo system, coupled with effective purification strategies, positions this as a promising platform for the scalable and cost-effective production of anellomid vectors for future therapeutic development. Compared to in vitro assembly methods, in vivo assembly demonstrates superior yield, significantly increasing anellomid concentration.

Optimization of the Anellomid System

Current and ongoing work focuses on optimizing the anellomid production system for enhanced yield, stability, and regulatory compliance.

Key Enhancements:

• Scaling production: Through high-density fermentation in bioreactors.

• Optimization strategies: To increase stability and yield of anellomids.

• Validating purification strategies: For downstream therapeutic use.

These optimizations collectively contribute to a more efficient and targeted gene delivery system, reinforcing the potential of the anellomid system for therapeutic applications.

Publications & Patents:

To be announced.