Research Terms
These adeno-associate vector capsid variants offer antibody-escaping abilities that will enable patients with antibodies against an AAV serotype to receive therapy or participate in clinical trials anyway. Due to the high incidence of target diseases and increased funding for gene therapy research, the market for AAV-based gene therapy is expected to grow exponentially with a growth rate of 43 percent in the period from 2019 to 2030. Up to 40 percent of the population has antibodies that neutralize AAV8, excluding them from AAV gene therapy or clinical trials utilizing AAV8. Other patients have neutralizing antibodies against AAVrh.10 and other AAV serotypes naturally or due to previous treatment with an AAV vector.
Researchers at the University of Florida have modified AAVrh.10 vectors to create improved variants that can evade neutralizing antibodies against different AAV serotypes that are structurally similar to AAVrh.10, most notably, AAV8. The variants will allow more patients previously treated with an AAV vector or those positive for neutralizing antibodies against AAVrh.10 and AAV8 to be treated using AAV gene therapy.
AAV capsid variants that evade neutralizing antibodies to AAVrh.10 and AAV8 in populations with antibodies to those serotypes, allowing more patients to receive gene therapy and participate in clinical trials.
The structure-guided approach by UF researchers aims to develop an AAVrh.10 vector capsid variant with tissue targeting and antibody escape abilities. Based on the analysis, the variant escapes recognition by antibodies cross-reactive to AAV8 and AAVrh.10, while maintaining the wild-type infectivity. This offers the opportunity to patients who have these antibodies for acceptance into clinical trials or treatments. Information of the receptor-binding site shows the possibility for AAV tissue tropism engineering, guiding the creation of altered tissue targets.
This panel of antibodies allows better characterization of antigenic epitopes on AAV8 and AAV9 capsids with the goal of mitigating patient immune response to the existing AAV based gene therapy. Adeno-associated viral (AAV) vectors are currently the tool-of-choice for gene therapy, due to their unique lifecycle and versatility in targeting different tissues. Some recent successful applications of AAV-based gene therapy include treatments for ophthalmologic and neurodegenerative diseases, among others. Although the use of AAV vectors is very promising, several challenges remain. One of the biggest challenges facing gene therapy is, overcoming host immune response to AAV-based gene delivery. Estimates claim that approximately 40-70 percent of the general population has been pre-exposed to AAVs and carries antibodies against AAV. ??Researchers at the University of Florida have developed a panel of monoclonal antibodies against AAV8 and AAV9 to facilitate characterization of their capsid-antibody interactions. AAV8 and AAV9 have become the choice vectors in targeting liver and central nervous system, but some immunogenicity has been observed in human and animal studies. The panel of antibodies against AAV8 and AAV9 developed at the University is an excellent tool for identification of potential mutation sites to allow next generation AAV vectors to evade the immune response and improve the efficacy of gene therapy.
Application
• Can be used to determine antigenic regions on the capsids of AAV8 and AAV9
• Potential to use in an ELISA tittering kit
• Potential to use in an immuno-dot blot assay
• Can be used in a neutralization assay
• These antibodies can be coupled to chromatography resins for improved binding and purification of AAV8 and AAV9 over current chromatography resins
Advantages
• To design a vector that can escape this host immune response has been an ongoing challenge in the field of gene therapy. This panel of monoclonal antibodies can be used as a tool to study the antigenicity of the AAV8 and AAV9 capsids
• These antibodies can also aid in characterizing variants being developed for improved therapeutic efficacy
• AAV capsid epitope of each antibody has been mapped
Technology
Using existing hybridoma technology, UF researchers generated two anti-AAV8 (designated HL2381 and HL2383; HL stands for Hybridoma Lab) and four anti-AAV9 (HL2368, HL2370, HL2372, and HL2374) IgG antibodies. The HL2372 antibody cross-reacts with AAV8. The clones were selected from fused hybridoma hybrids after capsid ELISA and native dot immunoblot screenings. All the six antibodies generated only bound to intact capsids, not the linear viral proteins, when tested. The identified isotypes of the two new anti-AAV8 antibodies, HL2381 and HL2383, are both IgG3. The identified isotypes of the four anti-AAV9 antibodies, HL2368, HL2370, HL2372, and HL2374, are IgG3, IgG2a, IgG2a, and IgG3, respectively. The two anti-AAV8 antibodies, HL2381 and HL2383, were able to recognize AAV8 as predicted, but also cross-reacted with AAV3B. During the clonal selection, one anti-AAV9 clone, HL2372, exhibited binding to both AAV8 and AAV9. This cross-reactivity was confirmed by dot immunoblot assay. All other anti-AAV9 antibodies, HL2368, HL2370, and HL2374, bound to AAV9 only.
These engineered AAV vectors are designed to target neuronal cell types to aid in the development of gene therapies, which can treat a variety of neurological diseases. In 2020, the market for CNS targeting technologies was worth $6 billion and is expected to reach $46.5 billion by 2030. Neurological diseases can result in life-threatening complications or lead to serious symptoms and/or death. Effective treatments remain limited. Gene therapies have the potential to treat individuals suffering or at risk for suffering from various neurological diseases and conditions.
Researchers at the University of Florida developed improved adeno-associated virus (AAV) vectors to target the central nervous system (CNS) for treatment of neuronal diseases and disorders.
AAV capsid modification that targets the central nervous system and other neuronal cell types to treat diseases that affect neurological tissue and/or function
University of Florida researchers have generated AAV capsids of the serotypes AAV1, AAV9 and AAVrh.10 containing a specific CNS-targeting protein insertion and have shown them to be effective at targeting different neuronal cell types, increasing transduction efficiency as much as 25 fold in certain circumstances. The insertion of the CNS targeting peptide also eliminates an immunogenic epitope, making the vectors less likely to be neutralized by the immune system.
This gene transfer vector helps locate and destroy malignant breast cancer cells that have spread to the bone marrow by delivering tumor-suppressing genes or cytotoxic drugs to wherever they are needed in the patient’s body. In about 25 percent of breast cancers, a protein called HER2/neu is found at 100 times normal levels. This protein encourages cell growth, causing HER2/neu-overexpressing cancers to be particularly aggressive. The only drug that targets HER2/neu-positive cancer is Herceptin®, which is ineffective against metastasis into the bone marrow. Approximately 30 percent of breast cancer patients eventually develop bone marrow metastasis, and no targeted treatment exists for these individuals. Researchers at the University of Florida have engineered parvovirus B19 into a form that easily targets HER2/neu-positive cancer cells and can deliver various types of therapy, including drugs and beneficial genes. The $8.6-billion-dollar breast cancer market is expected to reach $10.9 billion by 2018. Herceptin® (trastuzumab), the best-selling breast cancer drug, generates $3.4 billion in annual revenue alone, indicating the market for this gene transfer vector is sizable.
A gene transfer vector for treating HER2/neu-overexpressing cancer metastases that have spread to the bone marrow
Gene vectors are vehicles, often viruses, that can transport genetic material into living organisms when one or more of their original genes are faulty or missing. University of Florida researchers have inserted certain sequences into the gene coding of parvovirus B19’s capsid to develop a gene transfer vector that specifically targets HER2/neu-positive cells. The modified virus can bind to cells high in HER2/neu. In this way, the treatment is localized to only those cells high in HER2/neu, limiting damage to healthy tissue. The vector can transport therapeutic agents and proteins, reporter proteins used to detect presence of cancer cells, tumor-suppressor genes, suicide genes and cytotoxic drugs.
