Biology subjects

Khvorova, A.

Publications and source records attributed to Khvorova, A..

32 records · Page 2Linked to original sources

bioRxiv2023

A programmable dual-targeting di-valent siRNA scaffold supports potent multi-gene modulation in the central nervous system

Di-valent short interfering RNA (siRNA) is a promising therapeutic modality that enables sequence-specific modulation of a single target gene in the central nervous system (CNS). To treat complex neurodegenerative disorders, where pathogenesis is driven by multiple genes or pathways, di-valent siRNA must be able to silence multiple target genes simultaneously. Here we present a framework for designing unimolecular "dual-targeting" di-valent siRNAs capable of co-silencing two genes in the CNS. We reconfigured di-valent siRNA - in which two identical, linked siRNAs are made concurrently - to create linear di-valent siRNA - where two siRNAs are made sequentially attached by a covalent linker. This linear configuration, synthesized using commercially available reagents, enables incorporation of two different siRNAs to silence two different targets. We demonstrate that this dual-targeting di-valent siRNA is fully functional in the CNS of mice, supporting at least two months of maximal target silencing. Dual-targeting di-valent siRNA is highly programmable, enabling simultaneous modulation of two different disease-relevant gene pairs (e.g., Huntingtons disease: MSH3 and HTT; Alzheimers disease: APOE and JAK1) with similar potency to a mixture of single-targeting di-valent siRNAs against each gene. This work potentiates CNS modulation of virtually any pair of disease-related targets using a simple unimolecular siRNA.

molecular biology↗

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↗

bioRxiv2023

Dual targeting of hepatocyte DGAT2 and stellate cell FASN alleviates nonalcoholic steatohepatitis in mice.

Nonalcoholic steatohepatitis (NASH) is a malady of multiple cell types associated with hepatocyte triglyceride (TG) accumulation, macrophage inflammation, and stellate cell-induced fibrosis, with no approved therapeutics yet available. Here, we report that stellate cell fatty acid synthase (FASN) in de novo lipogenesis drives the autophagic flux that is required for stellate cell activation and fibrotic collagen production. Further, we employ a dual targeting approach to NASH that selectively depletes collagen through selective stellate cell knockout of FASN (using AAV9-LRAT Cre in FASNfl/fl mice), while lowering hepatocyte triglyceride by depleting DGAT2 with a GalNac-conjugated, fully chemically modified siRNA. DGAT2 silencing in hepatocytes alone or in combination with stellate cell FASNKO reduced liver TG accumulation in a choline-deficient NASH mouse model, while FASNKO in hepatocytes alone (using AAV8-TBG Cre in FASNfl/fl mice) did not. Neither hepatocyte DGAT2 silencing alone nor FASNKO in stellate cells alone decreased fibrosis (total collagen), while loss of both DGAT2 plus FASN caused a highly significant attenuation of NASH. These data establish proof of concept that dual targeting of DGAT2 plus FASN alleviates NASH progression in mice far greater than targeting either gene product alone.

molecular biology↗

bioRxiv2023

Extended Nucleic Acid (exNA): A Novel, Biologically Compatible Backbone that Significantly Enhances Oligonucleotide Efficacy in vivo

Metabolic stabilization of therapeutic oligonucleotides requires both sugar and backbone modifications, where phosphorothioate (PS) is the only backbone chemistry used in the clinic. Here, we describe the discovery, synthesis, and characterization of a novel biologically compatible backbone, extended nucleic acid (exNA). Upon exNA precursor scale up, exNA incorporation is fully compatible with common nucleic acid synthetic protocols. The novel backbone is orthogonal to PS and shows profound stabilization against 3- and 5-exonucleases. Using small interfering RNAs (siRNAs) as an example, we show exNA is tolerated at most nucleotide positions and profoundly improves in vivo efficacy. A combined exNA-PS backbone enhances siRNA resistance to serum 3-exonuclease by [~]32-fold over PS backbone and >1000-fold over the natural phosphodiester backbone, thereby enhancing tissue exposure ([~]6-fold), tissues accumulation (4- to 20-fold), and potency both systemically and in brain. The improved potency and durability imparted by exNA opens more tissues and indications to oligonucleotide-driven therapeutic interventions.

biochemistry↗

bioRxiv2023

Lactate transporter MCT1 in hepatic stellate cells promotes fibrotic collagen expression in nonalcoholic steatohepatitis

Circulating lactate is a fuel source for liver metabolism but may exacerbate metabolic diseases such as nonalcoholic steatohepatitis (NASH). Indeed, haploinsufficiency of lactate transporter monocarboxylate transporter 1 (MCT1) in mice reportedly promotes resistance to hepatic steatosis and inflammation. Here, we used adeno-associated virus (AAV) vectors to deliver thyroxin binding globulin (TBG)-Cre or lecithin-retinol acyltransferase (Lrat)-Cre to MCT1fl/fl mice on a choline deficient, high fat NASH diet to deplete hepatocyte or stellate cell MCT1, respectively. Stellate cell MCT1KO (AAV-Lrat-Cre) attenuated liver type 1 collagen protein expression and caused a downward trend in trichrome staining. MCT1 depletion in cultured human LX2 stellate cells also diminished collagen 1 protein expression. Tetra-ethylenglycol-cholesterol (Chol)-conjugated siRNAs, which enter all hepatic cell types, and hepatocyte-selective tri-N-acetyl galactosamine (GN)-conjugated siRNAs were then used to evaluate MCT1 function in a genetically obese NASH mouse model. MCT1 silencing by Chol-siRNA decreased liver collagen 1 levels, while hepatocyte-selective MCT1 depletion by AAV-TBG-Cre or by GN-siRNA unexpectedly increased collagen 1 and total fibrosis without effect on triglyceride accumulation. These findings demonstrate that stellate cell lactate transporter MCT1 significantly contributes to liver fibrosis through increased collagen 1 protein expression in vitro and in vivo, while hepatocyte MCT1 appears not to be an attractive therapeutic target for NASH.

molecular biology↗

bioRxiv2023

Self-delivering CRISPR RNAs for AAV Co-delivery and Genome Editing in vivo

Guide RNAs offer programmability for CRISPR-Cas9 genome editing but also add challenges for delivery. Chemical modification, which has been key to the success of oligonucleotide therapeutics, can enhance the stability, distribution, cellular uptake, and safety of nucleic acids. Previously, we engineered heavily and fully modified SpyCas9 crRNA and tracrRNA, which showed enhanced stability and retained activity when delivered to cultured cells in the form of the ribonucleoprotein complex. In this study, we report that a short, fully stabilized oligonucleotide (a "protecting oligo"), which can be displaced by tracrRNA annealing, can significantly enhance the potency and stability of a heavily modified crRNA. Furthermore, protecting oligos allow various bioconjugates to be appended, thereby improving cellular uptake and biodistribution of crRNA in vivo. Finally, we achieved in vivo genome editing in adult mouse liver and central nervous system via co-delivery of unformulated, chemically modified crRNAs with protecting oligos and AAV vectors that express tracrRNA and either SpyCas9 or a base editor derivative. Our proof-of-concept establishment of AAV/crRNA co-delivery offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation.

molecular biology↗

bioRxiv2022

Formamide significantly enhances the efficiency of chemical adenylation of RNA sequencing ligation adaptors

Pre-adenylated single-stranded DNA ligation adaptors are essential reagents in many next generation RNA sequencing library preparation protocols. These oligonucleotides can be adenylated enzymatically or chemically. Enzymatic adenylation reactions have high yield but are not amendable to scale up. In chemical adenylation, Adenosine 5{square}-phosphorimidazolide (ImpA) reacts with 5' phosphorylated DNA. It is easily scalable but gives poor yields, requiring labor-intensive cleanup steps. Here, we describe an improved chemical adenylation method using 95% formamide as the solvent, which results in the adenylation of oligonucleotides with >90% yield. In standard conditions, with water as the solvent, hydrolysis of the starting material to adenosine monophosphate limits the yields. To our surprise, we find that rather than increasing adenylation yields by decreasing the rate of ImpA hydrolysis, formamide does so by increasing the reaction rate between ImpA and 5'-phosphorylated DNA by [~]10 fold. The method described here enables straightforward preparation of chemically adenylated adapters with higher than 90% yield, simplifying reagent preparation for NGS.

molecular biology↗

bioRxiv2022

Di-valent siRNA Mediated Silencing of MSH3 Blocks Somatic Repeat Expansion in Mouse Models of Huntington's Disease

Huntingtons Disease (HD) is a severe neurodegenerative disorder caused by expansion of the CAG trinucleotide repeat tract in the huntingtin gene. Inheritance of expanded CAG repeats is needed for HD manifestation, but further somatic expansion of the repeat tract in non-dividing cells, particularly striatal neurons, hastens disease onset. Called somatic repeat expansion, this process is mediated by the mismatch repair (MMR) pathway. Among MMR components identified as modifiers of HD onset, MutS Homolog 3 (MSH3) has emerged as a potentially safe and effective target for therapeutic intervention. Here, we identify fully chemically modified short interfering RNA (siRNA) that robustly silence MSH3 in vitro and in vivo. When synthesized in a di-valent scaffold, siRNA-mediated silencing of MSH3 effectively blocked CAG repeat expansion in striatum of two HD mouse models without impacting tumor-associated microsatellite instability. Our findings establish a novel paradigm for treating patients with HD and other repeat expansion diseases. One Sentence SummarySilencing MSH3 in the CNS of two models of Huntingtons disease using di-valent siRNA blocks disease-accelerating somatic expansion of CAG repeats.

molecular biology↗

bioRxiv2022

Asymmetric trichotomous data partitioning enables development of predictive machine learning models using limited siRNA efficacy datasets

Chemically modified small interfering RNAs (siRNAs) are promising therapeutics guiding sequence-specific silencing of disease genes. However, identifying chemically modified siRNA sequences that effectively silence target genes is a challenge. Such determinations necessitate computational algorithms. Machine Learning (ML) is a powerful predictive approach for tackling biological problems, but typically requires datasets significantly larger than most available siRNA datasets. Here, we describe a framework for applying ML to a small dataset (356 modified sequences) for siRNA efficacy prediction. To overcome noise and biological limitations in siRNA datasets, we apply a trichotomous (using two thresholds) partitioning approach, producing several combinations of classification threshold pairs. We then test the effects of different thresholds on random forest (RF) ML model performance using a novel evaluation metric accounting for class imbalances. We identify thresholds yielding a model with high predictive power outperforming a simple linear classification model generated from the same data. Using a novel method to extract model features, we observe target site base preferences consistent with current understanding of the siRNA-mediated silencing mechanism, with RF providing higher resolution than the linear model. This framework applies to any classification challenge involving small biological datasets, providing an opportunity to develop high-performing design algorithms for oligonucleotide therapies.

bioinformatics↗

bioRxiv2022

Silencing of ApoE with Divalent siRNAs Drives Activation of Immune Clearance Pathways and Improves Amyloid Pathology in Mouse Models of Alzheimer's Disease

The most common genetic risk factor for late-onset Alzheimers disease (AD) is the APOE4 allele, with evidence for gain- and loss-of-function mechanisms. ApoE knockout in mice abrogates AD phenotypes but causes severe atherosclerosis due to the role of liver ApoE in cholesterol homeostasis. Previous attempts to inhibit brain-specific ApoE with anti-sense oligonucleotides only modestly reduced ApoE expression and had no effect on amyloid burden in adult AD mice. Here, we optimized a divalent small interfering RNA (di-siRNA) to selectively and potently silence ApoE in the brain. Silencing brain ApoE in AD mice significantly reduced amyloid plaque formation without affecting systemic cholesterol levels, confirming that brain and liver APOE pools are spatially and functionally distinct. Mechanistically, APOE appears to be a scaffold for beta-amyloid aggregation that limits clearance by microglia. Di-siRNAs from this study can be taken to pre-clinical and clinical trials to accelerate development of AD-modifying therapies and establish siRNA-based modulation of ApoE as a viable path towards therapeutic development.

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↗

bioRxiv2018

Diverse lipid conjugates for functional extra-hepatic siRNA delivery in vivo

RNAi-based therapeutics show promising clinical data for treatment of liver-associated disorders. However, siRNA delivery into extra-hepatic tissues remains an obstacle, limiting the use of siRNA-based therapies. Here we report on a first example of chemical engineering of lipophilic conjugates to enable extra-hepatic delivery. We synthesized a panel of fifteen lipophilic siRNA and evaluated the impact of their chemical configuration on siRNA tissue distribution profile. Generally, lipophilic conjugates allow siRNA distribution to a wide range of tissues, where the degree of lipophilicity defines the ratio of liver/spleen to kidney distribution. In addition to primary clearance tissues, several conjugates achieve significant siRNA distribution to lung, heart, adrenal glands, fat, muscle. siRNA tissue accumulation leads to productive silencing, shown with two independent targets. siRNA concentrations necessary for productive silencing are tissue and conjugate dependent, varying significantly from 5 to 200 ng/mg. The collection of conjugated siRNA described here enables functional gene modulation in vivo in lung, muscle, fat, heart, adrenal glands opening these tissues for future therapeutic intervention.

biochemistry↗

bioRxiv2018

Hydrophobicity drives the systemic distribution of lipid-conjugated siRNAs via lipid transport pathways

Efficient delivery of therapeutic RNA is the fundamental obstacle preventing its clinical utility. Lipid conjugation improves plasma half-life, tissue accumulation, and cellular uptake of small interfering RNAs (siRNAs). However, the impact of conjugate structure and hydrophobicity on siRNA pharmacokinetics is unclear, impeding the design of clinically relevant lipid-siRNAs. Using a panel of biologically-occurring lipids, we show that lipid conjugation modulates siRNA hydrophobicity and governs spontaneous partitioning into distinct plasma lipoprotein classes in vivo. Lipoprotein binding influences siRNA distribution by delaying renal excretion and promoting uptake into lipoprotein receptor-enriched tissues. Lipid-siRNAs elicit mRNA silencing without causing toxicity in a tissue-specific manner. Lipid-siRNA internalization occurs independently of lipoprotein endocytosis, and is mediated by siRNA phosphorothioate modifications. Although biomimetic lipoprotein nanoparticles have been considered for the enhancement of siRNA delivery, our findings suggest that hydrophobic modifications can be leveraged to incorporate therapeutic siRNA into endogenous lipid transport pathways without the requirement for synthetic formulation.

pharmacology and toxicology↗