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Generalizable and Non-genetic Approach to Create Metabolically-active-but-non-replicating Bacteria

Researchers at the University of California, Davis have developed a method to stop bacterial growth while maintaining desirable metabolic functions for therapeutic and biotechnological applications.

(2022-099) Repeat expansion disease therapy with antisense RNA vectors

Alternative splicing accounts for a considerable portion of transcriptomic diversity, as most protein-coding genes are spliced into multiple mRNA isoforms. However, errors in splicing patterns can give rise to mis-splicing with pathological consequences, such as the congenital diseases familial dysautonomia, Duchenne muscular dystrophy, and spinal muscular atrophy. Small nuclear RNA (snRNA) components of the U snRNP family have been proposed as a therapeutic modality for the treatment of mis-splicing. U1 snRNAs offer great promise, with prior studies demonstrating in vivo efficacy, suggesting additional preclinical development is merited. Improvements in enabling technologies, including screening methodologies, gene delivery vectors, and relevant considerations from gene editing approaches justify further advancement of U1 snRNA as a therapeutic and research tool.

Combinatorial Drug Therapy for Treating Resistant Glioblastoma Multiforme

Researchers at the University of California, Davis have discovered a new treatment target to attack and kill GBM cancer cells that evades macrophage phagocytosis and are resistant to radioimmunotherapy.

Epigenetic Prevention and Treatment of CDKL5 Deficiency Disorder

Researchers at the University of California, Davis have developed a targeted epigenetic approach for the prevention and treatment CDKL5 deficiency disorder.

CRISPR-Cas Effector Polypeptides and Methods of Use Thereof

CRISPR-Cas systems comprise a CRISPR-associated (Cas) effector polypeptide and a guide nucleic acid. Such CRISPR-Cas systems can bind to and modify a targeted nucleic acid. The programmable nature of these CRISPR-Cas effector systems has facilitated their use as a versatile technology for use in, e.g., gene editing.   UC Berkeley researchers have discovered new CRISPR-Cas effector Cas12L/Cas Lambda/Casλ polypeptides and methods of modifying a target nucleic acid using a Cas12L/Cas Lambda polypeptide.

Compositions and Methods for Treating Viral Infections

Researchers at the University of California, Davis (“UC Davis”) have developed methods for screening and targeting regions of viral genomes to identify drugs that inhibit the replication of RNA viruses.

Methods and Compositions for the Treatment of Huntington's Disease

There are no approved disease-modifying therapies for Huntington’s disease (HD), a fatal neurodegenerative condition caused by a heterozygous expansion of a CAG array in exon 1 of Huntingtin (Htt). Typically, HD patients are heterozygous for the toxic gain of function disease allele, yet expression of the wildtype version of the gene is essential. The inventors have developed methods and compositions to selectively silence expression from the disease-associated allele while leaving the wildtype version intact. The invention relies on the introduction of a 'poison' exon into the diseased allele wherein introduction of the poison exon may be accomplished by standard methods in the art, such as introduction of the exon sequences through homology-directed repair following targeted nuclease cleavage, transposon-associated targeted sequence introduction, base editing, and prime editing. Following the introduction of the poison exon, post-transcriptional splicing results in an RNA that is susceptible to nonsense mediated decay due to the introduction of a stop codon in the introduced exon. RNAs comprising the poison exon are subsequently degraded in the cell, effectively silencing expression of the mutant disease-associated allele.

COMPOSITIONS AND METHODS FOR REDUCING RNA LEVELS

Human diseases that follow a dominant negative inheritance pattern present a great challenge for treatment using gene therapy methods. In such cases, a copy of an allele is inherited from each parent: one is a pathogenic allele causing a disease phenotype (e.g., by exerting a toxic, gain-of-function effect) and the other is a wild-type (non-pathogenic) allele. Allele-specific targeting is especially important when the wild-type allele is crucial to normal function, e.g., the wild-type allele encodes a protein whose function is critical. There is therefore a need for compositions and methods of allele-specific gene editing.   UC Berkeley researchers have created methods and systems for reducing the level of an RNA transcript from a target nucleic acid in an allele-specific manner. Such systems and methods can be used to treat a disease that results from or is caused by a toxic gain-of-function protein.   

Type III CRISPR-Cas System for Robust RNA Knockdown and Imaging in Eukaryotes

Type III CRISPR-Cas systems recognize and degrade RNA molecules using an RNA-guided mechanism that occurs widely in microbes for adaptive immunity against viruses. The inventors have demonstrated that this multi-protein system can be leveraged for programmable RNA knockdown of both nuclear and cytoplasmic transcripts in mammalian cells. Using single-vector delivery of the S. thermophilus Csm complex, RNA knockdown was achieved with high efficiency (90-99%) and minimal off-targets, outperforming existing technologies of shRNA- and Cas13-mediated knockdown. Furthermore, unlike Cas13, Csm is devoid of trans-cleavage activity and thus does not induce non-specific transcriptome-wide degradation and cytotoxicity. Catalytically inactivated Csm can also be used for programmable RNA-binding, which the inventors exploit for live-cell RNA imaging. This work demonstrates the feasibility and efficacy of multi-subunit CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes.

Membrane-Associated Accessory Protein Variants Confer Increased AAV Production

The inventors have developed an engineering approach to identify novel and nonobvious membrane-associated accessory protein (MAAP) sequence variants that confer increased Adeno-associated virus (AAV) secretion during packaging. The technique is based upon the iterative process of sequence diversification and selection of functional gene variants known as directed evolution. First, the inventors generated a library of more than 1E6 MAAP variants. The variants were subjected to five rounds of packaging into an AAV2 capsid for which MAAP expression was inactivated without altering the viral protein VP1 open reading frame (ORF) (AAV2-MAAP-null). Among each iterative packaging round, the inventors observed a progressive increase in both the overall titer and ratio of secreted vector genomes conferred by the bulk selected MAAP library population. Next-generation sequencing uncovered common mutational features that were enriched up to over 10,000-fold on the amino acid level. Individual MAAP variants were isolated and systematically tested for effect on recombinant AAV2-MAAP-null packaging in HEK293 cells. The inventors predict that this work may be applicable to increasing per-cell AAV output in industrial settings, potentially reducing global costs and increasing functional vector recovery in downstream manufacturing processes.BACKGROUNDParvoviruses are small, single-stranded DNA viruses that are ubiquitously found in many animal species. AAV is a prototypic dependoparvovirus whose replication cycle requires the function of helper genes from larger co-infected viruses such as Adenoviruses or Herpesviruses. The natural genome of AAV contains ~4.7 kb of ssDNA that encodes up to ten known proteins in a highly overlapped fashion. The rep gene encodes four protein products named based on their molecular weight: Rep72 and Rep68 facilitate genomic replication, whereas Rep52, and Rep40 play essential roles in loading nascent ssDNA genomes into assembled capsids. Downstream of rep lies the cap gene, which encodes three known protein products off of overlapping reading frames: VP1, VP2, and VP3 are structural proteins that assemble to form the capsid, the assembly activating protein (AAP) targets VP proteins to the nucleus and is involved in capsid assembly. The most recently discovered AAV-encoded gene is the membrane-associated accessory protein (MAAP). MAAP is encoded by an alternative ORF in the AAV cap gene that is found in all presently reported natural serotypes. Gene delivery by recombinant AAV (rAAV) have shown significant success in both research and clinical gene therapy applications. In the rAAV system, Rep and Cap are removed from between AAV’s 5’ and 3’ inverted terminal repeats (ITRs) and provided in trans. Instead, a transgene of interest is inserted between the ITRs and subsequently packaged into the nascent AAV capsids. However, manufacturing quantities of good manufacturing practice (GMP)-grade rAAVs necessary to achieve current and projected dosing requirements–particularly in a clinical context–presents a significant hurdle to expanding rAAV-based gene therapies. Recently, evidence has emerged supporting a functional role of MAAP in AAV egress. This led to the hypothesis that MAAP could be engineered to facilitate increased levels of secreted AAV produced from HEK293 cells. 

Genetically Engineered Dendritic Cell-Derived Vaccines

Researchers at the University of California, Irvine have developed a new vaccine which generates a targeted, specific immune response with fewer complications than currently available vaccines.

Acid Degradable Solid Lipid Nanoparticles

The inventors demonstrate that polyethylene glycol (PEG) conjugated to cholesterol via an acid degradable linkage composed of an azide-benzaldehyde acetal has the potential to allow solid lipid nanoparticles (SLNs) to be PEGylated with mole ratios up to 50%. The azide-benzaldehyde acetal, has its azide in the para position, and generates stable acetals with a t ½ of > 1000 minutes at pH 7.4. These PEG-acetals can be formulated into SLNs, and stored, and then reduced prior to biological use, to generate an amino acetal that has t ½ < 60 minutes at pH 7.4 and several minutes at pH 5.0. The ultra-PEGylated lipids were efficient at transfecting a variety of organs, including the muscle, the lung, spleen and liver and were also able to transfect the blood. Acid degradable PEG-lipids have great potential for overcoming the PEG dilemma, but have previously been challenging to develop due to the synthetic challenges associated with working with acetals and their instability at pH 7.4. (SLNs contain a PEGylated lipid, generally in the 1-5% range, which is needed to maintain SLN stability, size, and tissue diffusion, and lower toxicity. However, excessive PEGylation also results in lower cell uptake and endosomal disruption — a paradox referred to as the PEG dilemma.) The inventors anticipate numerous applications of the azide-benzaldehyde acetal linker, given its unique ability to be stable prior to reductive activation. 

Multiplex Epigenetic Editing using a Split-dCas9 System

Researchers at the University of California, Davis have developed a new epigenetic editing system that overcomes packaging limitations of viral delivery systems and can be used for multiplexed epigenetic editing of a genome.

A Gene Therapy for treating Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC)

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a predominantly genetic-based heart disease characterized by right but also recently left ventricular dysfunction, fibrofatty replacement of the myocardium leading to fatal/severe ventricular arrhythmias leading to sudden cardiac death in young people and athletes. ARVC is responsible for 10% of sudden cardiac deaths in people ≥65 years of age and 24% in people ≤30 years of age. ARVC is thought to be a rare disease as it occurs in 1 in 1000-5000 people, although the prevalence may be higher as some patients are undiagnosed or misdiagnosed due to poor diagnostic markers. Growing evidence also reveals earlier onset since pediatric populations ranging from infants to children in their teens are also particularly vulnerable to ARVC, highlighting the critical need to identify and treat patients at an earlier stage of the disease. At present there are no effective treatments for ARVC nor has there been any randomized clinical trials conducted to examine treatment modalities, screening regimens, or medications specific for ARVC. As a result, treatment strategies for ARVC patients are directed at symptomatic relief of electrophysiological defects, based on clinical expertise, results of retrospective registry-based studies, and the results of studies on model systems. The current standard of care is the use of anti-arrhythmic drugs (sotalol, amniodarone and beta-blockers) that transition into more invasive actions, which include implantable cardioverter defibrillators and cardiac catheter ablation, if the patient becomes unresponsive or intolerant to anti-arrhythmic therapies. However, current therapeutic modalities have limited effectiveness in managing the disease, 40% of ARVC patients (a young heart disease) die within 10-11 years after initial diagnosis, highlighting the need for development of more effective therapies for patients with ARVC.

Treatment Of Inherited Retinal Disease

Researchers at UCI have developed a method of treating inherited retinal diseases, such as Leber congenital amaurosis (LCA) and retinitis pigmentosa, by gene therapy of the RPE65 nonsense mutation. This method uses base editor-mediated genome-editing by viral delivery and lead to improved patient treatment through enhanced editing of single base pairs and reduced off-target genomic editing.

Intranasal Delivery of Oligonucleotides for Neurodegenerative Diseases

Delivery of oligonucleotide therapy to the central nervous system remains challenging. Neurodegenerative diseases, such as Huntington’s disease and Spinal Muscular Atrophy, can require intrusive and regular treatments, therefore a non-invasive delivery system would be very beneficial to patients. UC Irvine researchers have proposed a new method of therapeutic delivery utilizing a SARS-CoV-2 pseudovirus. Delivered intranasally, this system has the ability to bypass the blood brain barrier, making it an exciting approach to decrease risk for patients and ease the treatment process.

Novel CRISPR-Cas Enzyme Variants and Methods of Use

CRISPR-Cas systems include Cas proteins, which are involved in acquisition, targeting and cleavage of foreign DNA or RNA, and a guide RNA(s), which includes a segment that binds Cas proteins and a segment that binds to a target nucleic acid. For example, Class 2 CRISPR-Cas systems comprise a single Cas protein bound to a guide RNA, where the Cas protein binds to and cleaves a targeted nucleic acid. The programmable nature of these systems has facilitated their use as a versatile technology for use in modification of target nucleic acid.   UC Berkeley researchers have discovered novel CRISPR-Cas proteins related to other CRISPR-Cas systems that utilize a single guide RNA (sgRNA) or a combination of a tracrRNA + guide RNA to perform RNA-directed cleavage of nucleic acids that can be applicable for DNA editing and diagnostics. The enzyme can cleave the target DNA and may be used for diagnostics by utilizing its ability to cleave single-stranded DNA in trans.  

(SD2019-040) Directed modification of cellular RNA via nuclear delivery of CRISPR/Cas

Present strategies aimed to target and manipulate RNA in living cells mainly rely on the use of antisense oligonucleotides (ASO) or engineered RNA binding proteins (RBP). Although ASO therapies have been shown great promise in eliminating pathogenic transcripts or modulating RBP binding, they are synthetic in construction and thus cannot be encoded within DNA. This complicates potential gene therapy strategies, which would rely on regular administration of ASOs throughout the lifetime of the patient. Furthermore, they are incapable of modulating the genetic sequence of RNA. Although engineered RBPs such as PUF proteins can be designed to recognize target transcripts and fused to RNA modifying effectors to allow for specific recognition and manipulation, these constructs require extensive protein engineering for each target and may prove to be laborious and costly. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin-top:0in; mso-para-margin-right:0in; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0in; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

Bioengineered Wnt5a Therapeutics For Advanced Cancers

Researchers at the University of California, Davis have developed RNA-based therapeutics to treat Wnt5A-expressing cancers, including treatment-resistant prostate cancer.

Natural Killer Cells with Enhanced Activity (SD 2021-141)

NK cells possess a native ability to kill tumors and virally infected cells without prior antigen priming. Furthermore, NK cells can be administered to patients across HLA allotypes, unlike T cells which require HLA matching to avoid graft-versus-host disease. Many trials utilizing adoptive transfer of allogeneic NK cells demonstrated complete remissions in patients with acute myelogenous leukemia (AML) who are refractory to standard chemotherapy. Another recent clinical study demonstrated effective treatment of lymphoid malignancies using allogeneic CAR-expressing NK cells, with minimal side effects. Thus, NK cells possess a number of advantages over T cells that enables them to be used as safe, effective, “off-the-shelf” adoptive cell therapy product to treat diverse malignancies. Antibody-dependent cellular cytotoxicity (ADCC) is a key pathway that mediates natural killer (NK) cell cytotoxicity against antibody-opsonized target cells. This process helps mediate the therapeutic efficacy of anti-tumor antibodies. On NK cells, ADCC occurs via engagement of antibody-coated target cells with activating receptor leading to proinflammatory cytokine upregulation, degranulation, and target cell death. Upon cellular activation, the     is cleaved from the NK cell surface. Cleavage of the ectodomain prevents further antibody binding and signaling, which dampens NK cell activity. Blocking activation-induced cleavage has been previously demonstrated to augment ADCC activity and provides a novel strategy to improve efficacy of therapeutic antibodies in combination with adoptive transfer of engineered NK cells. 

Engineered/Variant Hyperactive CRISPR CasPhi Enzymes And Methods Of Use Thereof

The CRISPR-Cas system is now understood to confer bacteria and archaea with acquired immunity against phage and viruses. CRISPR-Cas systems consist of Cas proteins, which are involved in acquisition, targeting and cleavage of foreign DNA or RNA, and a CRISPR array, which includes direct repeats flanking short spacer sequences that guide Cas proteins to their targets.  Class 2 CRISPR-Cas are streamlined versions in which a single Cas protein bound to RNA is responsible for binding to and cleavage of a targeted sequence. The programmable nature of these minimal systems has facilitated their use as a versatile technology that is revolutionizing the field of genome manipulation.  There is a need in the art for additional Class 2 CRISPR/Cas systems (e.g., Cas protein plus guide RNA combinations).     UC Berkeley researchers discovered a new type of CasPhi/12j protein.  Site-specific binding and/or cleavage of a target nucleic acid (e.g., genomic DNA, ds DNA, RNA, etc.) can occur at locations (e.g., target sequence of a target locus) determined by base-pairing complementarity between the Cas12 guide RNA (the guide sequence of the Cas12 guide RNA) and the target nucleic acid.  Similar to CRISPR Cas9, the compact Cas12 enzymes are expected to have a wide variety of applications in genome editing and nucleic acid manipulation.  

Improved guide RNA and Protein Design for CasX-based Gene Editing Platform

The inventors have developed two new CasX gene-editing platforms (DpbCasXv2 and PlmCasXv2) through rationale structural engineering of the CasX protein and gRNA, which yield improved in vitro and in vivo behaviors. These platforms dramatically increase DNA cleavage activity and can be used as the basis for further improving CasX tools.The RNA-guided CRISPR-associated (Cas) protein CasX has been reported as a fundamentally distinct, RNA-guided platform compared to Cas9 and Cpf1. Structural studies revealed structural differences within the nucleotide-binding loops of CasX, with a compact protein size less than 1,000 amino acids, and guide RNA (gRNA) scaffold stem. These structural differences affect the active ternary complex assembly, leading to different in vivo and in vitro behaviors of these two enzymes.

High Efficiency Single Cell Indexing Of Droplets Via Interfacial Shearing With Downstream Droplet Sorting

The invention is an integrated device that provides a high efficiency single cell encapsulation solution. The two core modules of the invention are responsible for generating the cell encapsulating droplet, then sorting the generated droplets to eliminate the empty ones. Such a two-step process yields a high throughput, single cell indexed droplets, with an overall encapsulation efficiency reaching 80%, which is crucial for various applications ranging from genomics and proteomics to pharmacology.

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