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The strategy employed by the invention is inspired by splicing factors, a category of RNA-binding protein that influence alternative splicing outcomes. These splicing factors are trans-acting, and act to enhance or silence exon inclusion by binding near or on the target exon and promoting or repressing the activity of splicing machinery. Scientifically, a highly programmable, minimally disruptive system to increase exon inclusion could allow for higher-throughput identification of functional roles of specific exons than have been previously shown.

Researchers at the University of California, Davis have developed a virally derived homolog to increase the inflammatory response desirable in cancer immunotherapy.

Researchers at the University of California, Davis have developed a viral peptide therapeutic that targets MYC-based cancerous tumors.

A primary reason behind the lack of progress in heart therapeutics is the inability to use phenotypic human tissue-level approaches to discover novel therapies. In recent years, there have been significant advances in the development microphysiological systems (MPS), which recapitulate organ-level and even organism-level functions.   MPS are quickly becoming representative of the future of disease modeling and drug screening, therefore paving the way for complex in vitro models to dominate the preclinical drug discovery landscape. However, there has yet to be an effective LNP formulation for therapeutic mRNA delivery to the heart. Therefore, despite progress in this area, one of the remaining challenges is to develop a LNP formulation capable of diffusing within human cardiac muscle, transfecting cardiomyocytes, and escaping the endo-lysosome before degradation more efficiently than current strategies. UC Berkeley researchers and others have developed compositions and methods using lipid nanoparticles for delivery of a payload (e.g., messenger RNA (mRNA)) to the heart, for delivery of mRNA for transfection of cells and methods of treatment.

Researchers at UC Irvine have identified and tested a molecular target that regulates T cell function during chronic viral infection and cancer. The molecular target is one of the high mobility group proteins (HMGB2). HMGB2 is a DNA binding protein that regulates transcriptional processes, meaning that its modulation will have profound effects on T cell differentiation and ultimate function by altering the expression of many genes.

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RNA-mediated adaptive immune systems in bacteria and archaea rely on Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) genomic loci and CRISPR associated (Cas) proteins that function together to provide protection from invading viruses and plasmids. Genome editing can be carried out using a CRISPR-Cas system comprising a CRISPR-Cas effector polypeptide and a guide nucleic acid, such as a guide RNA. However, unintended chromosomal abnormalities following on-target genome editing, such as chromosome loss, are potential concerns for genome editing. UC Berkeley researchers and others have developed a method to modulate the expression levels of the DNA damage response factor p53 in order to mitigate chromosomal abnormalities that occur after genome editing by nucleases like Cas9. The invention provides treatment methods by generating a modified cell and then administering the modified cell to an individual in need thereof and compositions having a CRISPR-Cas effector polypeptide, a guide nucleic acid, and an agent that increases the level of a p53 polypeptide in a mammalian cell.

Scientists have developed a CRISPR-Cas9 based genome editing method for universal correction of disease-causing mutations in the CTLA4 gene, which most commonly manifest as a Primary Immunodeficiency. Current treatment involves monthly IV injections or weekly subcutaneous injections of a recombinant CTLA4-Ig fusion protein abatacept. This invention includes one-time infusion of a CTLA4-corrected autologous T cell therapy. The corrected patient cells are generated by ex vivo electroporation of a specific gRNA:Cas9 ribonucleoprotien (RNP) complex and cognate homology-directed-repair template (HDRT) targeting a functional copy of the CTLA4 gene within an intronic region of the endogenous CTLA4 gene. This combination allows for (1) highly efficient knockin (up to 70% in patient cells), (2) cell-type and context specific regulation of CTLA4 expression under natural promoter and regulatory elements, and (3) preservation of endogenous CTLA4 expression in uncorrected cells.

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.

Manipulation of eukaryotic genomes, particularly the integration of multi-kilobase DNAsequences, remains challenging and limits the rapidly growing fields of synthetic biology andcell engineering. Large serine recombinases (LSRs) are enzymes that recognize specific targetsequences on a DNA donor sequence and DNA target sequence to catalyze a recombinationreaction that results in the insertion of a DNA donor in a sequence-specific manner. Genomeediting can be carried out using an LSR system and a DNA donor nucleic acid, such as a plasmidor double-stranded DNA. However, in a human genome, these systems can exhibit variableefficiency and specificity.In this invention, UC Berkeley researchers and others have developed optimized compositionsto significantly increase the efficiency and specificity of LSRs to target a specific genomic locusin human cells. Via fusion to additional protein systems, this engineered composition retainsthe simplicity of a single protein for gene delivery. The invention also encompasses use in invivo or ex vivo gene therapy and the creation of modified cell lines or transgenic animals.

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. The programmable nature of these 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 CRISPR-Cas systems with improved cleavage and manipulation under a variety of conditions and ones that are particularly thermostable under those conditions. UCB researchers created a set of efficient CRISPR-Cas9 proteins from a thermostable Cas9 from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9) through directed evolution. The gene editing activity of the evolved mutant proteins was improved by up to four orders of magnitude compared to the wild-type GeoCas9. The researchers showed that the gene editors based on the evolved GeoCas9 can be effectively assembled into lipid nanoparticles (LNP) for the rapid delivery to different cell lines in vitro as well as different organs or tissues in vivo. The LNP-based delivery strategy could also be extended to other gene editors.  

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.

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

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.

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.

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 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.

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. 

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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.

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.

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.