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Salmonella-Based Gene Delivery Vectors and their Preparation

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.

Diagnostic and Screening Methods for Atopic Dermatitis

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.

Chronic Villus Derived Stem Cells for Autologous Prenatal Therapy of Hemophilia A

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).

COMPOSITIONS AND METHODS FOR ENHANCED GENOME EDITING

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. 

Self-Inactivating Targeted DNA Nucleases For Gene Therapy

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.

A Transposon Vector From Aedes Aegypti For Use In Vertebrate And Invertebrate Gene Transfer

Background: Therapeutic delivery of genes is a rapidly evolving technique used to treat or prevent a disease at the root of the problem. Another widely used variation of this technique is to insert a transgene into animals and crops for production of desirable proteins. The global transgenic market is currently $24B with annual growth projections of 10%.  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 enhance current transgenic technologies as well as methods of gene therapy.

Improvements to Cas9-Mediated Mutation

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. 

Spinal Subpial AAV-mediated Gene or Antisense Oligonucleotide Delivery System

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.

NOVEL GENE THERAPY APPROACH TO TREATING LIVER FIBROSIS

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

Stabilizer Cells to Treat Cardiac Arrhythmias

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

MicroRNA-214 as a Diagnostic and Prognostic Biomarker for Ulcerative Colitis and Colitis-Associated Colon Cancer Patients

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.

Diagnostic, Prognostic and Therapeutic Uses of Non-Coding RNAs in Leukemia

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.

Dendritic Peptide Bolaamphiphiles for siRNA Delivery

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

Novel Chitosan Derivative as a Systemic Drug Delivery Agent and an Antibiotic Treatment

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.

Cost-effective Method to Quickly Produce and Purify Large Quantities of Biologically Active ncRNAs

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. 

Read-Through Compound Prodrugs Suppressing Premature Nonsense Mutations

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.

Dendronized Polymer Vectors For siRNA Delivery

Researchers at the University of California, Irvine have developed a new medium for delivery of siRNA genetic materials into cells. This medium is a vector, and its architecture allows for optimal siRNA binding. RNAi has tremendous potential for therapeutic treatment, and this vector allows for safe and efficient intracellular delivery of siRNA.

Novel Method of Using Modified and Optimized Bacterial-derived Genetic CRISPR System for Imaging, Regulating and Editing Mammalian Genomic Elements

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.

Autologous Adipose-Derived Mesenchymal Stem-Cell Therapy for Cats with Chronic Mucosal Inflammatory Disease

Chronic mucosal inflammatory diseases (for example, chronic gingivostomatitis) are poorly understood diseases in cats characterized by severe inflammation of the gums and oral cavity.  Cats with Chronic mucosal inflammatory disease have painful, debilitating lesions in their mouth that affect their ability to eat or be a suitable companion animal.  Unfortunately, , there is no truly effective medical treatment available, until now. Recently, researchers at the University of California, Davis have developed an innovative and fully effective treatment for this disease in cats by administering autologous Adipose-Derived Mesenchymal Stem-Cells (adMSC).

ULTRAHIGH THROUGHPUT (UHT) MICROINJECTION

Microinjection represents the “gold standard” for cellular manipulation, due to its precision, safety, and applicability to a wide variety of cell types and molecules.  However, the reliance of current instrumentation on skilled operators and serialized injection methodologies limits availability and throughput (~3 cells/min), thus hampering progress in many areas including ex-vivo cell therapies. Automation efforts have shown promise for improving success rates, but the expense of instrument complexity and limited gains in throughput (≤35 cells/min) have held back its universal adoption.    

Novel and Effective Gene Therapy for Critical Limb Ischemia

Critical limb Ischemia (CLI) represents a significant unmet medical need since there are currently no effective pharmaceuticals or biologic therapies for treatment of patients with occluded vessels. Researchers at the University of California, Davis have designed a Mesenchymal Stem/Stromal Cell (MSC) which secretes supraphysiological amounts of human Vascular Endothelial Growth Factor (VEGF) for revascularization of blood vessels and the treatment of peripheral artery diseases such as CLI.

Expression, Purification, And Isolation Of The Full Length Human Breast Cancer Susceptibility Gene 2 (Brca2) Protein

Method for expression, purification, and isolation of the full length human breast cancer susceptibility gene 2 (BRCA2) protein.

Cyclic Amp-Incompetent Adenylyl Cyclase Gene Transfer For Heart Failure

Heart failure is the most common cause of non-elective admission to the hospital in subjects 65 years and older. Despite optimal drug and device therapy, prognosis in heart failure is dismal. Many clinical trials of drugs that increase heart function (“inotropes”) have failed, possibly due to the deleterious effects of agents that increase cAMP. An alternative strategy is to alter myocardial calcium handling or myofilament response to calcium using agents that do not affect cAMP. Expression of a catalytically impaired adenylate cyclase type 6 mutant molecule (AC6mut), one that markedly reduces cAMP production, is associated with normal cardiac function in response to β-adrenergic receptor stimulation. The mechanism is through enhanced effects of AC6mut on Ca2+ handling - effects that do not require cAMP. These data are important in clinical settings for two reasons: 1) the results provide additional insight regarding the interplay between Ca2+ handling and βAR signaling vis-à-vis LV function; and 2) AC6mut may provide inotropic support free from the potentially deleterious effects of increased cAMP.

Treating Type 2 Diabetes by Targeting CAP Protein in the Macrophage

CAP (Cbl associated protein) is an adapter protein that is ubiquitously expressed. CAP acts in concert with Cbl to stimulate glucose uptake in skeletal muscle and adipose tissue as well as to induce the proliferation and migration of macrophages. Whole body CAP gene deletion in mice results in a protection from insulin resistance induced by high fat diet. However, exercise capacity is severely blunted in these mice.

Novel Pseudotyped Lentiviruses for Targeted Gene Therapy

UCLA researchers have developed a novel lentivirus pseudotype to specifically infect EphrinB2 expressing primary cells including difficult to transduce cell types like stem cells and neurons. This virus also has the ability to improve targeted gene therapy methods as it is the only known lentivirus not trapped in the liver following systemic delivery.

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