Biology subjects

Zin, E. A.

Publications and source records attributed to Zin, E. A..

6 recordsLinked to original sources

bioRxiv2026

Viability of engineered AAVs via protein language models

Capsid engineering has greatly improved the performance of recombinant AAV vectors used for gene therapy. One commonly used strategy is the insertion of a short, 7-mer, peptide into surface-exposed loops to modify receptor interactions and enhance cell entry. While effective in receptor retargeting and improved transduction, these insertions might destabilize the capsid protein, hinder assembly, and thus limit production. While previous attempts have used deep mutational scanning and AI to predict which insertions are viable, there is lack in understanding the structural consequences of these peptide insertions at the amino-acid level. Here we combined experiments, deep sequencing and large protein language models to gain insight on the impact of 7-mer insertions on the VR-VIII region. We first characterize the biochemical properties of viable insertions, thus identifying which residues are well tolerated, and which should instead be avoided. We then focus on the nearby context of those insertions, by studying the effect of the linkers, either for highly diverse libraries or for individual variants known for their efficiency. Next, we study the broader context, by extending our analysis to the whole capsid sequence, and identifying regions that can tolerate insertions without long-ranged structural deformations that could affect capsid functionality. We conclude with a cross-serotype comparison and a viability analysis of tens of previously engineered variants. Our work showcases how AI can uncover structure-function rules governing the success of engineered AAV capsids.

bioinformatics↗

bioRxiv2026

Promfusion: a synthetic fusion promoter enabling enhanced and balanced photoreceptor transgene expression

Achieving efficient and balanced transgene expression in both rods and cones remains a major challenge in retinal gene therapy. Current promoters either lack specificity or fail to provide sufficient cellular coverage and expression level. To address this limitation, we developed and evaluated two fusion promoters, Pikali and Nocchu, by combining PR1.7, a cone-specific promoter and GRK1, a promoter most active in rods. Here, we show that Pikali and Nocchu outperform their parental promoters, driving broader and more balanced GFP expression in rods and cones of human iPSC-derived retinal organoids. These constructs achieved transduction in 30% to 45% of photoreceptors, with higher expression levels than GRK1 and broader cellular coverage than PR1.7. Our findings establish Pikali and Nocchu as excellent candidates for retinal gene therapy, overcoming the limitations of existing promoters. By combining specificity, efficiency, and extensive photoreceptor targeting, these fusion constructs represent a novel and promising strategy for next-generation gene therapy vectors, addressing inherited retinal dystrophies and advancing clinical translation.

genetics↗

bioRxiv2024

The Role of Thermal Stability in AAV Titration of Engineered Variants

Determining the concentration of recombinant adeno-associated virus (AAV) productions, also known as titering, is crucial not only for quality control purposes but also for comparative studies of preclinical and clinical gene therapy trials. Recently, several AAVs were engineered by inserting seven amino acids at the outermost tip of the capsids protruding VR-VIII loop. These variants have demonstrated increased transduction capabilities over naturally occurring AAV serotypes in several studies. However, they have also been shown to produce lower yields when titered using standard techniques, raising questions about their adequacy for clinical development and use. Here, we investigated why peptide insertion onto AAV capsids reduces their titer by examining viral stocks using electron microscopy and PCR-based titering. We reveal that the DNAse digestion step, performed to eliminate free-floating DNA prior to qPCR or ddPCR, adversely impacts engineered capsid stability due to exposure to heat, artificially lowering viral titers of engineered serotypes. Titering without heating yields significantly higher titers for these variants which have melting temperatures (Tm) close to the DNAse inactivation temperature, while titers for parental serotypes with higher Tm remain unchanged. Our findings provide an important new perspective for titering engineered variants with lower thermostability, especially when comparing their effectiveness to their parental serotypes.

molecular biology↗

bioRxiv2024

Optimal sequencing depth for measuring the concentrations of molecular barcodes

In combinatorial genetic engineering experiments, next-generation sequencing (NGS) allows for measuring the concentrations of barcoded or mutated genes within highly diverse libraries. When designing and interpreting these experiments, sequencing depths are thus important parameters to take into account. Service providers follow established guidelines to determine NGS depth depending on the type of experiment, such as RNA sequencing or whole genome sequencing. However, guidelines specifically tailored for measuring barcode concentrations have not yet reached an accepted consensus. To address this issue, we combine the analysis of NGS datasets from barcoded libraries with a mathematical model taking into account the PCR amplification in library preparation. We demonstrate on several datasets that noise in the NGS counts increases with the sequencing depth; consequently, beyond certain limits, deeper sequencing does not improve the precision of measuring barcode concentrations. We propose, as rule of thumb, that the optimal sequencing depth should be about ten times the initial amount of barcoded DNA molecules before any amplification step.

bioinformatics↗

bioRxiv2023

In-silico monitoring of directed evolution convergence to unveil best performing variants with credibility score

Directed evolution (DE) is a versatile protein-engineering strategy, successfully applied to a range of proteins, including enzymes, antibodies, and viral vectors. However, DE can be time-consuming and costly, as it typically requires many rounds of selection to identify desired mutants. Next-generation sequencing allows monitoring of millions of variants during DE and can be leveraged to reduce the number of selection rounds. Unfortunately the noisy nature of the sequencing data impedes the estimation of the performance of individual variants. Here, we propose ACIDES that combines statistical inference and in-silico simulations to improve performance estimation in DE by providing accurate statistical scores. We tested ACIDES first on a novel random-peptide-insertion experiment and then on several public datasets from DE of viral vectors and phage-display. ACIDES allows experimentalists to reliably estimate variant performance on the fly and can aid protein engineering pipelines in a range of applications, including gene therapy.

bioinformatics↗

bioRxiv2021

Outcomes of Progranulin Gene Therapy in the Retina are Dependent on Time of Delivery

Neuronal ceroid lipofuscinosis (NCL) is a family of neurodegenerative diseases caused by mutations to genes related to lysosomal function. One variant, CNL11, is caused by mutations to the gene encoding the protein progranulin. Primarily secreted by microglia, progranulin regulates neuronal lysosomal function once endocytosed. Absence of progranulin causes cerebellar atrophy, seizures, ataxia, dementia and vision loss. As progranulin gene therapies targeting the brain are developed, it is also advantageous to focus on the retina, as its characteristics are beneficial for gene therapy development: the retina is easily visible through direct imaging, can be assessed through quantitative methods in vivo, requires smaller amounts of AAV and AAV can be administered via a less invasive surgery. In this study we characterize the retinal degeneration in a progranulin knockout mouse model of CLN11 and study the effects of gene replacement at different time points. All mice heterologously expressing progranulin showed reduction in lipofuscin deposits and microglia infiltration. While mice that receive systemic AAV9.2YF-scCAG-PGRN at post-natal day 3 or 4 show a reduction in retina thinning, mice injected intravitreally at months 1 and 6 with 7m8-scCAG-PGRN show no improvement, and mice injected at 12 months of age show increased retinal thinning in comparison to their controls. Thus, delivery of progranulin proves to be time-sensitive, requiring early administration for optimal therapeutic benefit.

neuroscience↗