Effects of Altering Heparan Sulfate Proteoglycan Binding and Capsid Hydrophilicity on Retinal Transduction by Adeno-associated Virus

ABSTRACT Adeno-associated viruses (AAVs) have recently emerged as the leading vector for retinal gene therapy. However, AAV vectors that are capable of achieving clinically relevant levels of transgene expression and widespread retinal transduction are still an unmet need. Using rationally designed AAV2-based capsid variants, we investigate the role of capsid hydrophilicity and hydrophobicity as it relates to retinal transduction. We show that hydrophilic, single-amino-acid mutations (V387R, W502H, E530K, L583R) in AAV2 negatively impact retinal transduction when heparan sulfate proteoglycan (HSPG) binding remains intact. Conversely, addition of hydrophobic point mutations to an HSPG binding-deficient capsid (AAV2ΔHS) leads to increased retinal transduction in both mouse and macaque. Our top performing vector, AAV2(4pMut)ΔHS, achieved robust rod and cone photoreceptor (PR) transduction in macaque, especially in the fovea, and demonstrates the ability to spread laterally beyond the borders of the subretinal injection (SRI) bleb. This study both evaluates biophysical properties of AAV capsids that influence retinal transduction and assesses the transduction and tropism of a novel capsid variant in a clinically relevant animal model. IMPORTANCE Rationally guided engineering of AAV capsids aims to create new generations of vectors with enhanced potential for human gene therapy. By applying rational design principles to AAV2-based capsids, we evaluated the influence of hydrophilic and hydrophobic amino acid mutations on retinal transduction as it relates to vector administration route. Through this approach, we identified a largely deleterious relationship between hydrophilic amino acid mutations and canonical HSPG binding by AAV2-based capsids. Conversely, the inclusion of hydrophobic amino acid substitutions on an HSPG binding-deficient capsid (AAV2ΔHS) generated a vector capable of robust rod and cone photoreceptor (PR) transduction. This vector AAV2(4pMut)ΔHS also demonstrates a remarkable ability to spread laterally beyond the initial subretinal injection (SRI) bleb, making it an ideal candidate for the treatment of retinal diseases that require a large area of transduction.

Hair Histology and Glycosaminoglycans Distribution Probed by Infrared Spectral Imaging: Focus on Heparan Sulfate Proteoglycan and Glypican-1 during Hair Growth Cycle

The expression of glypicans in different hair follicle (HF) compartments and their potential roles during hair shaft growth are still poorly understood. Heparan sulfate proteoglycan (HSPG) distribution in HFs is classically investigated by conventional histology, biochemical analysis, and immunohistochemistry. In this report, a novel approach is proposed to assess hair histology and HSPG distribution changes in HFs at different phases of the hair growth cycle using infrared spectral imaging (IRSI). The distribution of HSPGs in HFs was probed by IRSI using the absorption region relevant to sulfation as a spectral marker. The findings were supported by Western immunoblotting and immunohistochemistry assays focusing on the glypican-1 expression and distribution in HFs. This study demonstrates the capacity of IRSI to identify the different HF tissue structures and to highlight protein, proteoglycan (PG), glycosaminoglycan (GAG), and sulfated GAG distribution in these structures. The comparison between anagen, catagen, and telogen phases shows the qualitative and/or quantitative evolution of GAGs as supported by Western immunoblotting. Thus, IRSI can simultaneously reveal the location of proteins, PGs, GAGs, and sulfated GAGs in HFs in a reagent- and label-free manner. From a dermatological point of view, IRSI shows its potential as a promising technique to study alopecia.

Convergent chemoenzymatic synthesis and biological evaluation of a heparan sulfate proteoglycan syndecan-1 mimetic

A new convergent chemoenzymatic synthesis strategy has been established to efficiently synthesize a mimetic of structurally well-defined heparan sulfate proteoglycan syndecan-1 glyco-polypeptide at a milligram scale to enable biological studies.

Heparan sulfate proteoglycan 2 (HSPG2) is differentially expressed in metastatic breast cancer, both in metastases to the brain and to the lymph nodes.

Metastasis to the brain is a clinical problem in patients with breast cancer (1-3). We mined published microarray data (4, 5) to compare primary and metastatic tumor transcriptomes to discover genes associated with brain metastasis in patients with metastatic breast cancer. We found that heparan sulfate proteoglycan 2, also known as perlecan, encoded by HSPG2, was among the genes whose expression was most different in the metastatic tumor tissues of patients with metastatic breast cancer, both in metastases to brain and to the lymph nodes when compared to primary tumors of the breast or normal breast tissue, respectively. We observed significant down-regulation of HSPG2 in metastasis to the brain. Molecular functions and down-regulation of heparan sulfate proteoglycan 2 may be important for metastasis of primary tumor-derived cancer cells to the lymph nodes and to the brain in humans with metastatic breast cancer and suggests some level of common origin for metastases that reside in the lymph nodes and colonize the brain.