The remote control of cellular activation in a controllable and reproducible fashion is a key tool for biological research, as well as for therapeutic uses. Cellular therapies are becoming well established within the medical community. However, the degree of cellular activation can be an unknown factor, and the risk of off-target effects remains. Cells may be delivered, but may not be therapeutically effective, or effective cells may elicit activity in an undesired location. The delivery of a cell therapy where a known quantity of cell activation occurs at a specific, selected site may therefore be advantageous. UC San Diego researchers have recently developed the methods and materials for remote control of cellular activation, to dynamically manipulate molecular events for therapeutic effect.

The detection of levels of messenger RNA (mRNA), the molecule used by DNA to convey information about protein production, is a very important method in molecular biology. Current detection strategies, such as Northern Blotting and RT-PCR, require destruction of the cell to extract such information. Researchers at the University of California, Irvine have developed a method to non-destructively assess mRNA levels in a single living cell.

Background: Inflammatory Bowel Disease (IBD) is a chronic condition with substantial health and economic costs, affecting 1.3M people in the US. Currently, there is a lack of precise understanding of IBD and therefore, many are misdiagnosed or not even diagnosed at all. There is a high demand for effective and preventative therapies to reduce the burdens of IBD. Most of the time, patients have to resort to surgery which is a very expensive and invasive process. The IBD market is expected to be $9.6B in 2017 and is projected to show robust growth due to increasing IBD prevalence.   Brief Description: UCR researchers have discovered a novel gene that can be modulated to control the microbiome. It is also the first evidence of identifying a specific bacteria in a mouse model of human disease. This discovery will allow for insight into how and which human disease-associated genes are involved in modifying the microbiome to offer better therapeutic solutions in alleviating the disease.

Researchers at the University of California Davis have demonstrated that immature astrocytes generated from human pluripotent stems cells, promote oligodendrocyte lineage progression via TIMP-1 secretion.

Researchers at UCLA have developed a fusion protein that can detect immune cells expressing anti-CD19 chimeric antigen receptors with higher specificity and lower background than existing antibodies.

Many advancements to the Cas9 system (both the Cas9 nuclease and the sgRNA sequence) have been made to increase and optimize its efficiency and specificity.  Since many diseases and traits in humans have a complex genetic basis, multiple genomic targets must be simultaneously edited in order for diseases to be cured or for traits to be impacted.  Thus in order for CRISPR/Cas9 to be an effective gene therapeutic technology, huge swathes of the genome must be edited simultaneously, efficiently, and accurately. To address many of these issues, UC Berkeley researchers have developed a system method to rapidly manipulate multiple loci. This system allows for either sequential (maintaining inducible Cas9 present in the genome) or simultaneous (scarless excision) manipulation of Cas9 itself and can be applied to any organism currently utilizing the CRISPR technology.  The system can also be applied conveniently to create genomic libraries, artificial genome sequences, and highly programmable strains or cell lines that can be rapidly (and repeatedly) manipulated at multiple loci with extremely high efficiency.  

Nucleic acid-based gene interference technologies, including ribozymes and small interfering RNAs (siRNAs), represent promising gene-targeting strategies for specific inhibition of mRNA sequences of choice. A fundamental challenge to use nucleic acid-based gene interfering approaches for gene therapy is to deliver the gene interfering agents to appropriate cells in a way that is tissue/cell specific, efficient and safe. Many of the currently used vectors are based on attenuated or modified viruses, or synthetic vectors in which complexes of DNA, proteins, and/or lipids are formed in particles, and tissue-specific vectors have been only partially obtained by using carriers that specifically target certain cell types. As such, efficient and targeted delivery of M1GS sequences to specific cell types and tissues in vivo is central to developing this technology for gene targeting applications. Invasive bacteria, such as Salmonella, possess the ability to enter and transfer genetic material to human cells, leading to the efficient expression of transferred genes. Attenuated Salmonella strains have earlier been shown to function as a carrier system for delivery of nucleic acid-based vaccines and anti-tumor transgenes. Salmonella-based vectors are low cost and easy to prepare. Furthermore, they can be administrated orally in vivo, a non-invasive delivery route with significant advantage. Thus, Salmonella may represent a promising gene delivery agent for gene therapy. Scientists at UC Berkeley have developed a novel attenuated strain of Salmonella, SL101, which exhibited high gene transfer activity and low cytotoxicity/pathogenicity while efficiently delivering ribozymes, for expression in animals. Using MCMV infection of mice as the model, they demonstrated that oral inoculation of SL101 in animals efficiently delivered RNase P-based ribozyme sequence into specific organs, leading to substantial expression of ribozyme and effective inhibition of viral infection and pathogenesis. This strategy could easily be adopted deliver other gene targeting technologies.

Atopic dermatitis (AD) is a chronic itch and inflammatory disorder of the skin that affects one in ten people. Patients suffering from severe AD eventually progress to develop asthma and allergic rhinitis, in a process known as the “atopic march.” Signaling between epithelial cells and innate immune cells via the cytokine Thymic Stromal Lymphopoietin (TSLP) is thought to drive AD and the atopic march. TSLP is up regulated in atopic dermatitis patients and is thought to act on immune cells to trigger atopic dermatitis. Scientists at UC Berkeley discovered that TSLP also activates a subset of sensory neurons to signal itch by acting on TSLPR, which signals to TRPA1. They demonstrated that sensory neurons that transmit itch signals in AD are the only instance of signaling between TSLPR and TRPA1 in the same cell type. Therefore, blocking the signaling between TSLPR and TRPA1 is a novel and specific target for therapeutics for itch in atopic dermatitis. They also discovered that the Orai I/Stim I pathway triggers expression and secretion of TSLP. This pathway has never been directly demonstrated in human primary keratinocytes and has never before been linked to TSLP. Decreasing expression of Orai I or stim I using siRNA, or the downstream transcription factor, NFATc I, significantly attenuates TSLP secretion, as proven in mice studies. Thus inhibition of Orai I/Stim I/NFATc I signaling pathway is a novel target for therapeutics for itch in atopic dermatitis.

Researchers at the University of California, Davis have developed a method and composition using chorionic villus-derived stem cells that transgenically express Factor VIII for the treatment and prevention of hemophilia A (HA).

Genome editing holds great promise for fundamental discovery, treatment of genetic diseases, and prophylactic treatment.  Gene knockouts can be generated using a genome editing endonuclease (e.g., a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR/Cas protein: guide RNA, and the like) to introduce a site-specific double strand break (DSB) within a gene of interest.  Clones can be screened for those in which one or more alleles have been repaired in an error-prone fashion to disrupt the open reading frame.  However, genome editing reagents can have differential activities, for example variable knockout efficiency stemming from the use of different CRISPR guide RNAs.  Thus, there is a need for methods and compositions for increasing the frequency of disrupting mutations (e.g., indels) that can be produced when using targeted genome editing nucleases. UC Berkeley researchers have discovered a simple way to increase the frequency of the generation of indels gene editing reagents by adding non-homologous DNA to the genome targeting composition (e.g., zinc finger nuclease, TALEN nuclease fusion protein, CRISPR/Cas endonuclease). This approach greatly increases the frequency of knockout alleles, thereby enabling the easy generation of homozygous knockout cell lines and organisms, as well as improving the efficiency of knockout screens. 

The clinical application of targeted nucleases - such as zinc-finger nucleases, TALENs, and CRISPR/Cas9 – are exciting genome editing platforms. Delivery of nucleases to cells and tissues using as viral methods, however, can leave the nucleases stably present in the target cells, even after editing has been accomplished. One major safety concern is off-target effects (i.e. cutting a non-intended site), which pose a safety risk.  Another safety concern for gene therapies is the long-term expression of a foreign protein potentially provoking inflammatory reactions, another safety risk.   To avoid these potential detrimental outcomes, researchers at UC Berkeley have modified the delivered nuclease DNA which will cleave the host genome target DNA site and also excise its own DNA from the stable delivered construct.  The researchers have shown that there is no trace of any active delivered DNA remaining, thus mitigating the harmful side effects from nuclease based gene therapy.

Background: Therapeutic delivery of genes is a rapidly evolving technique used to treat or prevent a disease at the root of the problem. The global transgenic market is currently $24B, growing at an annual projected rate of 10%. Currently, a variation of this technique is widely used on animals and crops for production of desirable proteins, but this is a heavily infiltrated market. Thus, entering the gene therapy segment is more promising and would enhance the growth of this industry.  Brief Description: UCR Researchers have identified a novel transposon from Aedes aegypti mosquitoes. This mobile DNA sequence can insert itself into various functional genes to either cause or reverse mutations. They have successfully developed a transposon vector system that can be used in both unicellular & multicellular organisms, which can offer notable insight to improve current transgenic technologies as well as methods of gene therapy.

Cas9 is an RNA-guided DNA endonuclease used to perform targeted genomic manipulations, which can include the error-prone knockout of sequences via non-homologous end joining (NHEJ) and the introduction of precise edits via homology directed repair (HDR). HDR editing shows great promise for a variety of uses, such as generating new cellular immunotherapies, curing genetic disease, and introducing traits into agricultural crops. Yet the efficiency of HDR has lagged behind that of NHEJ, complicating these exciting applications. Additionally, worries have arisen about unintended knockout from off-target NHEJ.   UC Berkeley researchers have found that Cas9 operates by a surprising mechanism, which suggested ways to improve HDR. Taking advantage of this mechanism, researchers found simple methods to dramatically increase the efficiency of HDR, introducing targeted mutations in human cells with frequencies around 60%. Additionally, catalytically inactive Cas9 can be used to make mutations via HDR without attendant error-prone NHEJ. This latter activity allows the precise introduction of mutations with no danger of undesired knockout at off-target sequences. 

Currently used approaches to delivery vectors or ASO into the spinal parenchyma use one or two techniques, each having a substantial limitation as compared to this new delivery system. First, intrathecal delivery is used when vectors or ASO is injected into spinal intrathecal space (i.e. outside of pial membrane). Using this approach, no deep parenchymal transgene expression is seen after AAV9 delivery. Intrathecal delivery of ASO leads to a  good penetration of ASO into spinal parenchyma but is seen in the whole spinal cord (i.e. from cervical to sacral segments). No segment restricted distribution of ASO can be achieved by intrathecal delivery. A second technique, also in use currently, is a direct spinal parenchymal injection. By using this approach a segment specific transgene expression or ASO distribution can be achieved in spinal parenchyma. A major limitation of this technique is its invasive nature because a direct spinal parenchymal needle penetration is required.

This invention establishes a new approach to treating liver fibrosis using gene therapy.

UCLA researchers in the Department of Cardiology have developed a novel gene therapy for cardiac arrhythmias.

Dr. Dimitrios Iliopoulos in UCLA Department of Medicine has identified a novel biomarker, microRNA-214 (miR-214), that predicts, at near 100% specificity, an ulcerative colitis patient’s risk for developing colon cancer.

The Rao group at UCLA has developed a method of using lincRNA expression levels as a diagnostic and prognostic tool for B acute lymphoblastic leukemia. Furthermore, regulation of certain leukemia-associated lincRNA may hold therapeutic potential.

Cas9 is an endonuclease that binds complementary target DNA and generates site-specific breaks using two conserved nuclease domains. By inactivating both nuclease domains, dCas9 is produced, which functions as a programmable DNA binding protein. Current methods use dCas9-GFP fusions to image chromosomal loci, but have insufficient signal-to-noise ratio and often misidentify loci. UC Berkeley researchers have engineered a Cas9 variant that can be labeled with small molecule fluorescent dyes. This variant utilizes a conformational change in Cas9 to provide highly specific identification of chromosomal loci, and has been shown to work in a proof-of-principle experiment using Förster resonance energy transfer (FRET) pairs.

Novel dendritic peptide bolaamphiphiles that are safe and efficient for siRNA delivery.

RNA-programmed Cas9 has proven to be a versatile tool for genome engineering in multiple cell types and organisms.  Guided by a dual-RNA complex or a chimeric single-guide RNA, Cas9 (or variants of Cas9) can generate site-specific double-stranded DNA breaks (DSBs) or single-stranded breaks (SSBs) within target nucleic acids.  Target nucleic acids can include double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) as well as RNA.  When cleavage of a target nucleic acid occurs within a cell, the break in the target nucleic acid can be repaired by non-homologous end joining or homology directed repair.  UC Berkeley researchers have created new Cas9 protein variants, nucleic acids encoding the variant Cas9 proteins, and host cells comprising the nucleic acids.

Researchers at the University of California, Irvine have developed a novel chitosan derivative that may be used simultaneously as a systemic drug delivery agent and a systemic antibiotic treatment.

Researchers at the University of California, Davis have developed a novel method to biosynthesize large quantities of biologically active ncRNA agents (e.g., miRNAs and siRNAs) for functional and therapeutic uses. 

UCLA researchers in the Department of Neurology have identified a novel prodrug to enhance the aqueous solubility of RTC13 for the treatment of Duchenne Muscular Dystrophy and other genetic disorders caused by nonsense mutations.

This invention is a novel method using optimized small guide RNAs (sgRNAs) to enable dynamic imaging, editing and regulation of specific genomic elements in living mammalian cells via the CRISPR system.