How Are AAV Vectors Transforming Gene Therapy?

Adeno-associated virus (AAV) has emerged as a powerful tool in gene therapy, offering a safe and effective means of delivering therapeutic genes to target cells. Discovered in the 1960s, AAV is a small, non-enveloped virus that can infect human cells without causing disease, making it an ideal candidate for therapeutic applications. The blog below will delve into the fundamental aspects of AAV, its role in gene therapy, the benefits and challenges associated with its use, and ongoing advancements to enhance its effectiveness and speed up development.

What is AAV?

AAV is a small, non-enveloped virus that belongs to the Parvoviridae family. Discovered in the 1960s, AAV is notable for its ability to infect human cells without causing disease, making it an attractive candidate for gene therapy. The virus requires a helper virus (such as adenovirus or herpesvirus) to replicate, but AAV can remain latent within cells in its absence.

How AAV Works in Gene Therapy

Adeno-associated virus (AAV) works in gene therapy as a vector to deliver therapeutic genes into target cells. The process begins with designing AAV vectors, replacing viral genes with the desired therapeutic gene.¹ These engineered vectors are then produced in large quantities. Upon administration, AAV vectors enter the patient's cells and deliver the therapeutic gene into the nucleus. This gene is then expressed to produce the necessary proteins, potentially correcting the genetic defect. AAV is favored due to its safety, ability to achieve long-term gene expression, and versatility in targeting various tissues.

Primary Benefits of Using AAV for Gene Therapy Delivery

Vector Design 

Scientists modify AAV by removing its viral genes and replacing them with the therapeutic gene of interest. This modified AAV is incapable of causing disease.

Production 

Large quantities of the engineered AAV vectors are produced in laboratory settings using helper viruses or plasmids to facilitate replication.

Delivery

The AAV vectors are delivered to the patient's target cells through direct injection into tissues or intravenous administration.

Transduction

The AAV vectors enter the target cells and deliver the therapeutic gene into the cell's nucleus. The therapeutic gene can then be expressed, producing the desired protein and potentially correcting the genetic defect.

The Advantages of AAV Gene Therapy

AAV gene therapy offers several advantages. It is relatively safe due to AAV's non-pathogenic nature and low immunogenicity, reducing the risk of adverse immune responses. AAV vectors can achieve long-term expression of therapeutic genes, particularly in non-dividing cells, minimizing the need for repeated treatments. They have broad tropism, allowing them to target a wide range of tissues and organs. AAV vectors can also integrate stably at specific sites in the genome, providing persistent gene expression. These qualities make AAV a versatile and effective tool for treating various genetic disorders, offering hope for long-term disease management and potential cures.¹

Safety

AAV's non-pathogenic nature and low immunogenicity make it a safer option than other viral vectors.²

Long-Term Expression

AAV vectors can achieve long-term therapeutic gene expression, particularly in non-dividing cells, reducing the need for repeated treatments.²

Broad Tropism

Different AAV serotypes can target various tissues, allowing for versatile applications in various organs.²

Stable Integration

While AAV primarily remains episomal (outside the host genome), it can integrate at specific sites in the genome, providing stable and persistent gene expression.²

The Current Challenges With AAV Gene Therapy

Navigating AAV-based gene therapy involves tackling challenges in packaging, immune response, and tissue targeting. The limited packaging size of rAA) vectors and potential impurities increase design, production complexity, and costs. Additionally, immune responses and the need for precise gene expression control further complicate the delivery of effective and safe therapies.

AAV Packaging & Manufacturing

Generally, the packaging size of a recombinant AAV (rAAV) vector is limited to 5 kb (including the ITR sequences) and smaller. If vectors are larger than that, viral production yields are greatly reduced, and packaged vectors are truncated. Truncated rAAV constructs are non-therapeutic and dilute the purity (and potentially the safety) of rAAV preparations.³

Construct truncations and other impurities, such as chimeric vectors or host or helper plasmid DNA, can also arise due to unforeseen construct design choices. The generation of these non-therapeutic impurities during production can sometimes be caused by DNA secondary structure and the stalling of the replication machinery.⁴ With these contaminants comes the need for downstream enrichment, which can be technically challenging, expensive, and inefficient. This ultimately raises the overall cost of gene therapies that are approved for commercialization.⁵

Tissue Tropism

When systemically administered to humans, most natural AAVs accumulate in the liver and can efficiently transduce hepatocytes. However, different AAV serotypes enable delivery to various other tissues, helping drive specificity.²

Immune Response

Patients may have pre-existing immunity to AAV due to natural exposure, or they might develop an immune response after treatment, reducing the efficacy of the therapy.

Limited Cargo Capacity

AAV vectors have a limited capacity to carry genetic material, which makes treating diseases requiring larger or multiple genes challenging.

Manufacturing Complexity

Producing AAV vectors at the scale and consistency required for widespread clinical use is complex and costly.⁶

Gene Expression Control

Achieving precise control over the level and duration of gene expression remains difficult, which is crucial for effective and safe therapy.²

Insertional Mutagenesis

Although rare, there is a risk that AAV integration into the genome can disrupt other genes, potentially leading to unintended effects.⁷

Targeting Specific Tissues

While AAV can target a range of tissues, ensuring efficient and specific delivery to the desired cells in the body is still a challenge.

Improving Effectiveness and Accelerating Speed of AAV Gene Therapy Development

Overcoming the efficacy and safety challenges requires a multi-faceted approach focused on optimizing AAV constructs and capsids for the manufacturing scale-up beyond the small-scale production required in the preclinical stages.

Addressing AAV Immune Response

Recombinant DNA is nothing new, and regulatory elements and transgenes involved in construct design are often equated to interchangeable modules, where they can be put in different contexts and behave similarly.

With AAV-based gene therapies, there is significant variation in how different constructs can behave at every step of development and manufacturing. 

For instance, mRNA and protein expression can be challenging to predict and is highly dependent on the transduced cell types, secondary structure, metabolic state of the cell, and more. There’s also an enhanced risk of immunotoxicity associated with CpG islands, which may not affect the overall expression of the transgene but can cause significant safety issues.⁸

‍Partially filled AAV capsids have also been found to be a common impurity in AAV production runs.⁹ These can arise due to secondary structures and certain sequence motifs present in rAAV constructs. These can lead to AAV capsids being filled with partial constructs, which can ultimately lead to the production of an immunogenic truncated transgene or the need to administer high doses to reach desired levels of efficacy. 

Developing ways to perform multi-factorial optimization for all of these potential complications early in development can save significant time and effort for gene therapy developers. In silico solutions are currently in development to predict gene expression, compare multiple constructs across different parameters (e.g., yield, safety, and expression), packaging, truncation, and more.

Enhanced AAV Vector Design 

Optimizing construct design early in development to improve the quality and yield of manufactured AAV products is one way to design for manufacturing right from the start. In addition, using techniques that efficiently and comprehensively characterize AAV manufacturing runs can provide the data to further optimize manufacturing processes. One example is long-read sequencing, which offers a more complete understanding of construct variants (e.g. mutations, truncated sequences, etc.) within manufactured rAAV.

‍In silico technology also provides algorithms that enable AAV yield and quality predictions for bioreactor runs and provide improved AAV product characterization to ensure a thorough understanding of product composition. 

Scalable Manufacturing 

Significant effort has also been made to improve the tissue tropism or cell specificity of AAV serotypes. Rational design, directed evolution, and in silico approaches have been used to engineer capsids with improved transduction of muscle cells, lung alveoli, and retinal endothelial.^10,11

As AAVs have small packaging capacities, there have also been construct design efforts that minimize the size of the transgene and regulatory elements required for efficacy. In principle, AAV capsid engineering could also help increase the capacity of currently available AAVs.

Form Bio’s Partner Program to Advance AAV Gene Therapy Development

Form Bio offers powerful In Silico Assays^TM capabilities complimenting pre-clinical research to IND for AAV gene therapy development. This range of capabilities allows us to be a co-development partner with gene therapy companies in a broad, program-like manner from the early stages of drug design through preclinical development and regulatory approval to begin clinical development. 

We are widening our moat by securing and sequencing what we believe is the most comprehensive aggregation of high-fidelity NGS data on gene therapy constructs, with a variety of genes of interest, promoters, etc. and incorporating this data into our training and inference models.

AI Disclosure: Feature image was generated with AI-image tool, MidJourney.