# Novel strategy to block Nabs for AAV gene delivery

> **NIH NIH R01** · UNIV OF NORTH CAROLINA CHAPEL HILL · 2022 · $578,782

## Abstract

Adeno-associated virus (AAV) vectors have been applied in clinical trials in patients with different disorders.
Although successful in clinical studies, one of major concerns for broader AAV vector application for patients is
high prevalence of neutralizing antibody (Nab). In the general human population, over 95% of individuals have
been infected by AAV and, on average, approximate 50% of them have Nabs. Several approaches have been
explored to evade AAV Nabs, including epitope masking with PEG or exosome, different serotype of AAV
vector, rational design and combinatorial mutagenesis of the capsid in situ, as well as biological depletion of
Nabs (empty capsid utilization, B cell depletion, plasma-apheresis and IgG cleavage enzymes). Generally,
these approaches have low efficiency or side effects or AAV tropism change. Therefore, it is imperative to
develop ideal strategies to evade Nabs, but without a change in tissue tropism from capsid engineering or
negative side effects from pharmacological treatment. Recently, we have developed a vector independent
protein based strategy to universally block Nabs and demonstrated that this approach is effective against a
broad range of pre-existing Nab concentrations by use of a unique mycoplasma derived protein and it’s
analogues, termed Protein-M. Protein-M is able to interact with immunoglobulin from any species without
antigen dependence. We have found that protein M protected AAV vector neutralization over 100 fold in vitro
and 1000 fold in mice with adoptive transfer of Nab positive serum, so far, the most effective strategy to evade
AAV Nabs. However, the wild type protein M is structurally unstable at body temperature (37°C), making it
challenging for clinical application. The protein unfolding at body temperature may correspond with decreased
Nab blockade when using the protein in vivo. In order to improve PM stability, we have rationally designed in
silico a library of 150 individual protein mutants engineered for improved thermal stability. At least 10 mutants
were identified with enhanced thermal stability and high biological activity. In this proposal, we will further
characterize the efficacy of these mutants in vitro and in mice with adoptive transfer of Nab positive serum
(Aim 1). Next, we will study the role of protein M in AAV re-dosing in mice with pre-immunization (Aim 2). Then
we will move forward to a disease model to test the ability of the best protein M variants to block AAV Nabs in
animals for phenotypic correction with AAV vector mediated gene delivery (Aim 3). The long term objective is
to develop a more effective strategy to evade Nab activity in future gene therapy with AAV vectors in patients
with Nabs.

## Key facts

- **NIH application ID:** 10416627
- **Project number:** 1R01AI168242-01
- **Recipient organization:** UNIV OF NORTH CAROLINA CHAPEL HILL
- **Principal Investigator:** Chengwen Li
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $578,782
- **Award type:** 1
- **Project period:** 2022-03-01 → 2027-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10416627

## Citation

> US National Institutes of Health, RePORTER application 10416627, Novel strategy to block Nabs for AAV gene delivery (1R01AI168242-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10416627. Licensed CC0.

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