Gene therapy applications necessitate cell transfection techniques for delivering biomaterial into multiple or a single cell(s). The global market for transfection technologies can be worth more than half a billion by 2017. Current viral and chemical transfection techniques have limited ease of fabrication, transfection efficiency, dosage control, and cell viability. The invention discloses a simple yet efficient technique for nanoinjection of material into a single cell with high transfection efficiency, controlled dosage delivery, and full cell viability.

96 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:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; 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;} 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.     UC researchers discovered a new type of RNA-guided endonuclease (GeoCas9) and variants of GeoCas9.  GeoCas9 was found to be stable and enzymatically active in a temperature range of from 15°C to 75°C and has extended lifetime in human plasma.  With evidence that GeoCas9 maintains cleavage activity at mesophilic temperatures, the ability of GeoCas9 to edit mammalian genomes was then assessed.  The researchers found that when comparing the editing efficiency for both GeoCas9 and SpyCas9, similar editing efficiencies by both proteins were observed, demonstrating that GeoCas9 is an effective alternative to SpyCas9 for genome editing in mammalian cells.  Similar to CRISPR-Cas9, GeoCas9 enzymes are expected to have a wide variety of applications in genome editing and nucleic acid manipulation.   

Researchers at the University of California, Davis have discovered a class of compounds that both bind to a unique newly-discovered binding site in respiratory complex III and act as inhibitors of electron transport for use as mitochondrial anti-cancer drugs.

This invention identifies a novel class of natural protein-based inhibitors of CRISPR-Cas9, which could eliminate off-target effects of Cas9-mediated gene editing. It also presents an attractive antibiotic strategy and a potential biodefense agent against CRISPR bioterror threat.

UCLA researchers in the Department of Chemical and Biomolecular Engineering have developed a novel immunotherapeutic strategy that uses a selectively-activated cytotoxic molecule to enable tumor-specific T cell-mediated killing.

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 immunotherapeutic effect.

Researchers at UC Irvine developed an independent non-invasive method to distinguish between healthy and unhealthy embryos.

During seizures or pain-induced inflammation, excess chemical mediators suppress potassium channels mediating neuronal activity and thereby inactivate new generation anti-epileptic drugs and painkillers acting on those channels. The invention describes a gene therapy using a genetically-engineered potassium channel that reduces adverse effects by silencing neuronal hyperactivity while maintaining normal neuronal activity in the presence of chemical mediators to treat epilepsy and pain.

The obesity epidemic is an ongoing issue leading to significant economic and social burden, in part due to its role in the development of diabetes. Only three DFA-approved drugs for obesity treatment currently exist, none of which are without significant side effects and risks. Researchers at UCI have developed a DNA-based approach that activates metabolism, to target genes only in the fat and liver, causing increased energy expenditure and weight loss without affecting other organs. These present a viable approach to obesity treatment with minimal side effects in comparison to current drug treatments.

UCLA researchers in the Department of Microbiology, Immunology & Molecular Genetics have developed a method to permanently correct the out-of-frame dystrophin gene in patient cell models of Duchenne Muscular Dystrophy (DMD)

Neuropathic pain is a common problem, though, there are few existing pain medications have specific targets to treat this type of pain, and often lack efficacy and tolerance. The invention identifies specific proteins and related genes as targets for treating neuropathic pain in an animal model.

The invention consists of a multi-channel, droplet-generating microfluidic device with a strategically placed feature. The feature vibrates in order to counteract particle-trapping micro-vortices formed in the device. Counteracting these vortices allows for single particle encapsulation in the droplets formed by the device and makes this technology a good candidate for use in single cell diagnostics and drug delivery systems.

The Mikkola group at UCLA has discovered a novel regulator of hematopoietic stem cell self-renewal. The overexpression of this regulator increases the yield of ex vivo stem cell expansion and could thereby improve the efficiency of stem cell therapies. 

96 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:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Calibri; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin;} 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.  Current CRISPR Cas technologies are based on systems from cultured bacteria, leaving untapped the vast majority of organisms that have not been isolated.  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 Cas protein, CasY.  CasY is short compared to previously identified CRISPR-Cas endonucleases, and thus use of this protein as an alternative provides the advantage that the nucleotide sequence encoding the protein is relatively short.  CasY utilizes a guide RNA to perform double stranded cleavage of DNA. The researchers introduced CRISPR-CasY into E. coli, finding that they could block genetic material introduced into the cell.  Further research results indicated that CRISPR-CasY operates in a manner analogous to CRISPR-Cas9, but utilizing an entirely distinct protein architecture containing different catalytic domains.   CasY is also expected to function under different conditions (e.g., temperature) given the environment of the organisms that CasY was expressed in.  Similar to CRISPR Cas9, CasY enzymes are expected to have a wide variety of applications in genome editing and nucleic acid manipulation.   

96 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:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Calibri; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin;} 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.  Current CRISPR Cas technologies are based on systems from cultured bacteria, leaving untapped the vast majority of organisms that have not been isolated.  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 Cas protein, CasX, from groundwater samples. CasX is short compared to previously identified CRISPR-Cas endonucleases, and thus use of this protein as an alternative provides the advantage that the nucleotide sequence encoding the protein is relatively short.  CasX utilizes a tracrRNA and a guide RNA to perform double stranded cleavage of DNA. The researchers introduced CRISPR-CasX into E. coli, finding that they could block genetic material introduced into the cell.  Further research results indicated that CRISPR-CasX operates in a manner analogous to CRISPR-Cas9, but utilizing an entirely distinct protein architecture containing different catalytic domains.   CasX is also expected to function under different conditions (e.g., temperature) given the environment of the organisms that CasX was expressed in.  Similar to CRISPR Cas9, CasX enzymes are expected to have a wide variety of applications in genome editing and nucleic acid manipulation. 

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.

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.

Bacterial adaptive immune systems employ CRISPRs and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. Although generally targeted to DNA substrates, the Type VI CRISPR system directs interference complexes against single-stranded RNA substrates and in Type VI CRISPR systems, the single-subunit Cas13a/C2c2 protein functions as an RNA-guided RNA endonuclease.   UC Berkeley researchers have discovered that the CRISPR-Cas13a/C2c2 has two distinct RNase activities that enable both single stranded target RNA detection and multiplexed guide-RNA processing.  These dual RNase functions were found to be chemically and mechanistically different from each other and from the CRISPR RNA processing behavior of the evolutionarily unrelated CRISPR enzyme Cpf1.  Methods for detecting the single stranded target RNA were also discovered using a Cas13a/C2c2 guide RNA and a Cas13a/C2c2 protein in a sample have a plurality of RNAs as well as methods of cleaving a precursor Cas13a/C2c2 guide RNA into two or more Cas13a/C2c2 guide RNAs.  

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.

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.