Biology subjects

Alterman, J. F.

Publications and source records attributed to Alterman, J. F..

6 recordsLinked to original sources

bioRxiv2026

Fully Modified SpyCas9 Guide RNAs Enable Robust Genome Editing In Cells and In Vivo

Precision engineering of CRISPR/Cas9 components has advanced genome editing toward therapeutic applications. Completely chemically stabilized guide RNAs (gRNAs) have the potential to improve in vivo editing efficacy while enabling greater flexibility in delivery strategies. However, previous generations of fully modified guides have been associated with reduced Cas9 activity. Here, we employed an iterative, structure-guided optimization strategy to systematically introduce chemical modifications at each position of SpyCas9 gRNAs. Extending beyond commonly used nucleotide modifications, we incorporated 2-amino-RNA, 4-thio-RNA, and extended nucleic acid (exNA) to generate gRNA designs in which 90-100% of the nucleotides are sugar- or backbone-modified. Although certain modification patterns exhibit sequence-dependent variability, we have established a growing repertoire of guides that consistently maintain or enhance editing efficacy when applied both in vitro and in vivo. Collectively, our heavily and fully modified gRNAs hold potential for applications in nuclease editing, base editing, and other genome editing tools. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/725424v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1118f08org.highwire.dtl.DTLVardef@1c57069org.highwire.dtl.DTLVardef@1572148org.highwire.dtl.DTLVardef@14a111e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Divalent siRNA for prion disease

Prion protein (PrP) lowering is effective in animal models of prion disease and is being tested clinically in prion disease patients, but there remains a need for more potent PrP-lowering drug candidates. Inspired by the reported potency and duration of action of divalent short interfering RNA (siRNA), a new oligonucleotide drug modality for the central nervous system, we sought to discover and develop a new PrP-lowering drug candidate. Herein we identify a mouse Prnp-targeting divalent siRNA molecule, 1682-s4, that lowers PrP to 49% residual brain expression in wild-type mice, and, in the context of intracerebral infection with Rocky Mountain Laboratory (RML) prions, achieves a 2.7-fold increase in survival time with pre-symptomatic chronic treatment and 64% increase in survival time with a single dose after symptom onset. We describe the generation of two transgenic mouse lines, Tg25109 and Tg26372, expressing the full human PRNP gene and its non-coding sequence, and demonstrate their utility for in vivo discovery of potent human PRNP-targeting oligonucleotides. We discover siRNA sequence 2439 against human PRNP and compare its potency in different divalent siRNA chemical scaffolds. We determine that both the fixed UU tail and extended nucleic acid linkages of scaffold s4 contribute to superior potency compared to other scaffolds tested, offering 9.4 and 15.9 percentage points respectively of additional PrP knockdown. A single dose of 348 {micro}g of 2439-s4 lowered whole brain hemisphere human PrP in transgenic mice to 17% residual after 30 days, while 52 {micro}g lowered PrP to 49% residual. 1-2% of the dose of 2439-s4 delivered into cerebrospinal fluid is retained in the brain, and the median effective tissue concentration is estimated at 1.2 micrograms per gram of tissue. Good Laboratory Practices toxicology studies identified no significant liabilities, and the U.S. FDA has cleared an Investigational New Drug application to bring 2439-s4 into clinical trials.

neuroscience↗

bioRxiv2024

Potent and durable gene modulation in heart and muscle with chemically defined siRNAs

Small interfering RNA (siRNAs) hold immense promise for treating cardiac and muscular diseases, but robust and scalable delivery to these tissues remains a challenge. Recent advances in delivery strategies to muscle include conjugation of biologics (antibody/antibody fragments, peptides), which are currently in clinical development. However, the manufacturing of biologic-siRNA conjugates is a challenging and complex process. By contrast, lipophilic siRNAs are readily chemically synthesized at scale and support sufficient cardiac and skeletal muscle delivery. In this work, we refine siRNA design elements to enhance potency and durability and support clinically relevant silencing in muscle. Applying this strategy for siRNAs targeting myostatin (MSTN), a key target in muscle-wasting conditions, we show that a single subcutaneous dose in mice achieved robust and durable silencing ([~]80% inhibition up to 6 weeks, [~]30% at 14 weeks). Biweekly dosing resulted in >95% reduction of circulating MSTN for half a year, with no observed systemic or target-related toxicity. MSTN inhibition resulted in muscle growth and increased lean muscle mass, correlating with improved grip strength. Interestingly, the functional impact on muscle growth and strength significantly outlasts the target silencing, suggesting extended pharmacological effects. Systemic administration was equally efficacious in all muscle groups tested, including skeletal muscle, heart, tongue and diaphragm. The informational nature of the muscle-active chemically defined siRNA scaffold was confirmed by demonstrating muscle and heart efficacy with three additional targets. Our findings pave the way for potent and long-lasting gene modulation in muscle using chemically defined, lipophilic siRNAs, offering a new avenue for treating muscular diseases.

pharmacology and toxicology↗

bioRxiv2023

Single intravitreal administration of a tetravalent siRNA exhibits robust and efficient gene silencing in rodent and swine photoreceptors

Inherited retinal dystrophies caused by dominant mutations in photoreceptor-expressed genes, are a major cause of irreversible vision loss. Oligonucleotide therapy has been of interest in diseases that conventional medicine cannot target. In the early days, small interfering RNAs (siRNAs) were explored in clinical trials for retinal disorders with limited success due to a lack of stability and efficient cellular delivery. Thus, an unmet need exists to identify siRNA chemistry that targets photoreceptor-expressed genes. Here we evaluated 12 different fully chemically modified siRNA configurations, where the valency and conjugate structure were systematically altered. The impact on retinal distribution following intravitreal delivery was examined. We found that the increase in valency (tetravalent siRNA) supports the best photoreceptor accumulation. A single intravitreal administration induces multi-months efficacy in rodent and porcine retinas while showing a good safety profile. The data suggest that this configuration can treat retinal diseases caused by photoreceptor-expressed genes with 1-2 intravitreal injections per year.

neuroscience↗

bioRxiv2022

Chemical engineering of therapeutic siRNAs for allele-specific gene silencing in vivo in CNS

Small interfering RNAs (siRNAs) are a new class of drugs, exhibiting sequence-driven, potent, and sustained silencing of gene expression in vivo. We recently demonstrated that siRNA chemical architectures can be optimized to provide efficient delivery to the CNS. Many genetically-defined neurodegenerative disorders are autosomal dominant favoring selective silencing of the mutant allele. In some cases, successful targeting of the mutant allele requires targeting of a single nucleotide polymorphism (SNP) heterozygosity. Using Huntingtons disease as a model, we demonstrate allele-specific RNAi-based silencing of gene expression in vivo and in neurons differentiated from HD patient-derived iPSCs. A series of in vitro screens, with chemical and thermodynamic optimization, identified compounds with >50-fold selectivity for the mutant HD-causing allele, based on a single nucleotide difference. The optimized compound exhibits selective silencing of mutant huntingtin (HTT) protein in patient derived cells and throughout the HD mouse brain, providing a demonstration of SNP-based allele-specific RNAi silencing of gene expression in vivo in the CNS. The ability to target a disease-causing allele using RNAi-based therapies could be applied to a wide range of dominant CNS disorders, where maintenance of wild-type expression is essential.

neuroscience↗

bioRxiv2018

Heavily and Fully Modified RNAs Guide Efficient SpyCas9-Mediated Genome Editing

RNA-based drugs depend on chemical modifications to increase potency and nuclease stability, and to decrease immunogenicity in vivo. Chemical modification will likely improve the guide RNAs involved in CRISPR-Cas9-based therapeutics as well. Cas9 orthologs are RNA-guided microbial effectors that cleave DNA. No studies have yet explored chemical modification at all positions of the crRNA guide and tracrRNA cofactor. Here, we have identified several heavily-modified versions of crRNA and tracrRNA that are more potent than their unmodified counterparts. In addition, we describe fully chemically modified crRNAs and tracrRNAs (containing no 2-OH groups) that are functional in human cells. These designs demonstrate a significant breakthrough for Cas9-based therapeutics since heavily modified RNAs tend to be more stable in vivo (thus increasing potency). We anticipate that our designs will improve the use of Cas9 via RNP and mRNA delivery for in vivo and ex vivo purposes.

biochemistry↗