Skip to main content

Engineering AAV vectors for enhanced safety profiles

 Engineering AAV vectors for enhanced safety profiles involves multiple strategies at both the vector genome and capsid levels. Here is a breakdown of these strategies:



Vector Genome Level:

  • Modifying Vector Genome Sequences: Scientists modify AAV vector genomes by adding, mutating, or deleting specific sequences. For example, self-complementary AAV (scAAV) vectors are designed by deleting key signals from the second inverted terminal repeat (ITR), allowing for more efficient genome replication.
  • Codon Optimization: Optimizing the codon usage of the transgene can enhance its expression efficiency.
  • Promoter and PolyA Sequence Selection: Careful selection and manipulation of promoter and polyadenylation (polyA) sequences can influence transgene expression and tissue specificity.


Capsid Engineering:

Capsid engineering strategies can be categorized into four main categories:

  • Directed Evolution: This approach involves creating capsid mutant libraries using error-prone PCR or introducing peptides with known or speculated affinities into the capsid. It relies on selecting capsids that exhibit desired properties.
  • Rational Design: Scientists use knowledge of capsid structure and function to make precise modifications to improve capsid properties. This approach is guided by insights from structural studies.
  • Computer-Guided Design: Computational modeling and simulations help predict how specific capsid modifications may affect interactions with host cells and the immune system.
  • Combinations: Combining different strategies, such as directed evolution and rational design, can optimize capsids for specific purposes.


Circumventing Immune Responses:

AAV vectors, despite low immunogenicity, can still trigger immune responses that affect safety. Strategies to address these issues include:

  1. CpG Motif Modification: Unmethylated CpG motifs in AAV vectors can activate immune responses. Modifying the vector's genome to reduce CpG motifs can mitigate this risk.
  2. Innate Immunity Sensors: Understanding how AAV vectors interact with innate immunity sensors like TLR9 and MDA5 can help design vectors that minimize immune activation.
  3. Neutralizing Antibody Escape: Some strategies focus on escaping neutralizing antibodies (NAbs) to maintain vector efficacy. Directed evolution and rational design can generate AAV variants that are less susceptible to NAbs.
  4. Complement Activation: Complement activation can pose risks. Capsid engineering approaches aim to understand and modify AAV capsids to reduce complement activation, especially in the alternative pathway.


Minimizing Genotoxicity:

  • Integration Control: While AAV vectors typically do not integrate into the host genome, there is a low risk of genotoxicity. Scientists explore methods to control integration, including using homology arms for targeted integration and CRISPR-Cas9 combinations.
  • Partial Genomes: Strategies are devised to prevent the generation of partial genomes, which may lead to genotoxicity. Inverted genomes for self-complementary AAVs and deleting promoter-proximal D-sequences for single-stranded AAVs are considered.
  • Promoter and Enhancer Selection: The choice of promoters and enhancers can affect the risk of genotoxicity. Some regulatory elements may lead to overexpression or oncogenicity, while others can be used to limit transgene expression to safe levels.


Controlling On-Target Delivery and Off-Target Expression:

  • Tissue-Specific Promoters: Using tissue-specific promoters or regulatory elements ensures that gene expression is limited to the target tissue, reducing off-target effects.
  • Clonal Selection: Clonal selection techniques help identify AAV clones that exhibit the highest transduction efficiency in the target tissue while minimizing off-target transduction.
  • Balancing Safety and Efficacy: Striking the right balance between transduction efficiency and safety is crucial, especially when using high vector doses or systemic delivery.


In summary, engineering AAV vectors for improved safety profiles involves a multifaceted approach that encompasses vector genome modifications, capsid engineering, immune response mitigation, genotoxicity control, and precise control over on-target and off-target transduction. These strategies aim to enhance the safety and efficacy of AAV-based gene therapies.


For details

https://www.frontiersin.org/articles/10.3389/fmmed.2022.1054069/full

Popular posts from this blog

Human Genome Editing: FDA Draft Guidance Summary

Consideration for Developing Gene Editing Product  1. Genome Editing Methods: Genome editing can be achieved through nuclease-dependent or nuclease-independent methods. Nuclease-dependent methods involve introducing site-specific breaks in DNA using technologies like zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), modified-homing endonucleases, and CRISPR-associated (Cas) nucleases. These breaks can lead to modification of the DNA sequence at the cleavage site. Nuclease-independent methods can change DNA sequences without cleaving the DNA and include techniques like base editing and synthetic triplex-forming peptide nucleic acids. The choice of GE technology should consider factors such as the mechanism of action, the ability to target specific DNA sequences, and the potential to optimize components for efficiency, specificity, or stability. 2. Type and Degree of Genomic Modification: Different GE approaches rely on DNA repair pathways such a...
Read more

Stem loop RT-PCR for Detection of siRNA in Animal Tissues

Step Loop RT-PCR for Detection of Small Interfering RNA (siRNA) The recent publications described a novel used the novel method for the detection of siRNAs using a TaqMan®-based approach. This approach utilizes similar strategy that has been used for microRNA detection. The approach is illustrated in below.  In brief, the RT step occurs in the presence of a stem-loop RT primer that is complementary to the last 6–10 bases of the 3′ end of the antisense strand of the target siRNA. The stem-loop primer contains an additional universal sequence at the 5′ end that facilitates a TaqMan-based detection strategy in the subsequent qPCR step. As in the case of microRNA, the forward primer for qPCR is sequence-specific for the target siRNA. For sequence compositions that yield a low predicted melting temperature (Tm), the forward primer is designed as a tailed primer to help increase Tm. Stem Loop PCR for SiRNA Detection Step 1: Preparation of liver and plasma samples for the quanti...
Read more

Stem-Loop PCR for siRNA

 Stem-loop PCR is a method often used for detecting and quantifying small RNAs, such as siRNA or miRNA, which are typically difficult to amplify directly due to their short lengths. The method involves the design of a stem-loop reverse transcription (RT) primer, which enhances specificity and stability of the short RNA during the RT-PCR process, allowing for sensitive detection and quantification of the siRNA. Here’s a detailed guide to how stem-loop PCR can be applied to siRNA detection: Key Steps in Stem-Loop PCR for siRNA Designing the Stem-Loop RT Primer : Structure : The stem-loop RT primer consists of a loop region flanked by complementary sequences on either side (the "stem"), which will fold back on itself to form a hairpin structure. Specific Binding Region : A short sequence complementary to the 3’ end of the siRNA is added at the end of the stem-loop primer to ensure specific binding to the siRNA target. Stabilization : The loop structure helps prevent primer-dimer...
Read more

Allometric scaling in AAV gene therapy dose estimation

 Allometric scaling in AAV gene therapy dose estimation is crucial for translating effective and safe doses from animal models to humans. Since AAV dosing often involves high viral vector concentrations, proper dose scaling is essential to minimize adverse effects and optimize therapeutic outcomes. Here’s a breakdown of how allometric scaling is applied in AAV gene therapy dosing and the considerations involved: 1. Concept of Allometric Scaling Allometric scaling is a method of adjusting drug doses across species based on body size, physiology, and metabolism. It is especially useful in biologics and gene therapies where the pharmacokinetics and pharmacodynamics are more complex than small-molecule drugs. For AAV vectors, dosing is commonly scaled by body weight (e.g., vector genomes [vg] per kilogram) or body surface area (BSA), as these parameters can approximate dose distribution and vector exposure across different species. 2. Standard Scaling Approaches Body Weight Scaling (mg...
Read more

Cell-Mediated Immunity in AAV Gene Therapy

Cell-mediated immunity (CMI) plays a significant role in the effectiveness and safety of AAV (Adeno-Associated Virus) gene therapy. Understanding the impact of CMI is crucial for optimizing therapeutic outcomes and managing potential adverse effects. Here’s a detailed overview of the impact of CMI on AAV gene therapy: 1. Mechanisms of Cell-Mediated Immunity in AAV Gene Therapy T-Cell Activation : After administration of an AAV vector, T cells can recognize the AAV capsid proteins or the transgene product as foreign antigens, leading to their activation. This can involve both CD4+ helper T cells and CD8+ cytotoxic T cells. Cytokine Production : Activated T cells produce cytokines (e.g., IFN-γ, TNF-α) that can enhance the immune response. These cytokines can influence the activation and proliferation of other immune cells, including B cells and macrophages. 2. Impact on Efficacy of AAV Gene Therapy Enhanced Antigen Presentation : CMI can improve the presentation of transgene-derived anti...
Read more