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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. 

RNA-Guided Fusion Proteins for Targeted Diversification of Cytoplasmic DNA

The inventors have developed a method of mutagenizing user-defined regions of cytoplasmic DNA using a single guide RNA (sgRNA) or combinations of sgRNAs and a highly engineered fusion polypeptide comprising: a nuclear export sequence (NES)-containing, engineered nuclear localization sequence (NLS)-lacking, enzymatically active, RNA-guided endonuclease that introduces a single-stranded break in cytoplasmic DNA, and an error-prone DNA polymerase. This novel technology encompasses and provides evidence for the use of RNA-guided nucleases with relaxed PAM requirements, which are particularly useful for AT-rich targets such as the vaccinia virus genome. The inventors show that the truncation of up to several base pairs from the PAM-distal template binding region of the sgRNAs significantly increases the functional activity and specificity of the targeted mutagenesis complex. Moreover, the invention describes specific methods for the use of this technology to edit cytoplasmically replicating viruses with large DNA genomes, using poxviruses as a model system. The novel editing platform and methods selectively and continuously accelerate diversification of user-defined sites in the vaccinia genome during infection, while retaining most library members, due to significantly lowering deleterious off-target mutations. BACKGROUND Nucleocytoplasmic large DNA viruses (NCLDVs) are a group of viruses that harbor large (150 kbp - 1.2 mbp) double-stranded DNA genomes and replicate in the cytoplasm of eukaryotic cells. An example of an NCLDV that has historically been among the most prominent tools in human health is vaccinia, a poxvirus. Hundreds of millions of humans have been intentionally inoculated with vaccinia as part of a global effort to eliminate smallpox, which was declared eradicated in 1980.Vaccinia and some other poxviruses remain highly scientifically relevant in the post-eradication world. They are useful as vaccines against deadly poxvirus outbreaks that could potentially arise from natural spillover, bioterrorism, or biowarfare, as well as due to their therapeutic promise as oncolytic agents to selectively deliver anti-cancer transgenes and recruit adaptive immunity while leaving healthy cells unharmed. Directed evolution is a powerful engineering technique for evolving new phenotypes that are beneficial for biotechnological applications but for which there may have never been a selective pressure to evolve in nature. Both natural and directed evolution depend upon generation of genetic diversity, followed by a selective pressure. While natural evolution generates genetic diversity randomly and throughout the entirety of the genome, directed evolution ideally focuses mutations within specific genomic windows connected to the phenotype that one wishes to engineer. However, there is a need in the art for compositions and methods for mutagenizing a target DNA in the cytoplasm of mammalian cells. NCLDVs, which either partially or entirely express their own replicative and translational machinery independent of the nucleus, are difficult, and in many cases impossible, to produce from plasmid DNA in cells. Thus, NCLDVs are not amenable to standard in vitro molecular diversification strategies.  

Fumigant Detoxification via Reusable Cotton Material

Researchers at the University of California, Davis have developed wearable, highly adsorptive, cotton fabrics that can neutralize fumigants in both open-air and sequestered environments.

Protein Inhibitor of Type VI-B CRISPR-Cas System

The inventors have discovered the first protein inhibitor of the type VI-B CRISPR-Cas system. By controlling this CRISPR system, one could possibly ameliorate the toxicity and off-target cleavage activity observed with the use of the type VI CRISPR system. Moreover, these proteins can also serve as an antidote for instances where the use of CRISPR-Cas technology poses a safety risk. Additionally, this technology can also be used for engineering genetic circuits in mammalian cells. This finding is of potential importance to many companies in the CRISPR space. 

Deep Learning Techniques For In Vivo Elasticity Imaging

Imaging the material property distribution of solids has a broad range of applications in materials science, biomechanical engineering, and clinical diagnosis. For example, as various diseases progress, the elasticity of human cells, tissues, and organs can change significantly. If these changes in elasticity can be measured accurately over time, early detection and diagnosis of different disease states can be achieved. Elasticity imaging is an emerging method to qualitatively image the elasticity distribution of an inhomogeneous body. A long-standing goal of this imaging is to provide alternative methods of clinical palpation (e.g. manual breast examination) for reliable tumor diagnosis. The displacement distribution of a body under externally applied forces (or displacements) can be acquired by a variety of imaging techniques such as ultrasound, magnetic resonance, and digital image correlation. A strain distribution, determined by the gradient of a displacement distribution, can be computed (or approximated) from measured displacements. If the strain and stress distributions of a body are both known, the elasticity distribution can be computed using the constitutive elasticity equations. However, there is currently no technique that can measure the stress distribution of a body in vivo. Therefore, in elastography, the stress distribution of a body is commonly assumed to be uniform and a measured strain distribution can be interpreted as a relative elasticity distribution. This approach has the advantage of being easy to implement. The uniform stress assumption in this approach, however, is inaccurate for an inhomogeneous body. The stress field of a body can be distorted significantly near a hole, inclusion, or wherever the elasticity varies. Though strain-based elastography has been deployed on many commercial ultrasound diagnostic-imaging devices, the elasticity distribution predicted based on this method is prone to inaccuracies.To address these inaccuracies, researchers at UC Berkeley have developed a de novo imaging method to learn the elasticity of solids from measured strains. Our approach involves using deep neural networks supervised by the theory of elasticity and does not require labeled data for the training process. Results show that the Berkeley method can learn the hidden elasticity of solids accurately and is robust when it comes to noisy and missing measurements.

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.

Metabolic Assessment to Diagnose Equine Neuroaxonal Dystrophy (eNAD)/ Equine Degenerative Myeloencephalopathy (EDM)

Researchers at the University of California, Davis have developed a protocol and assay to assess the rate of metabolism of vitamin E in horses that serves as a potential diagnostic test for equine neuroaxonal dystrophy (eNAD) and equine degenerative myeloencephalopathy (EDM).

Immunogenic Composition to Prevent Francisella Infections in Fish

Researchers from the University of California, Davis are part of a multi-institution research team that has developed a vaccine with proven efficacy in preventing Francisella infections in fish.

Compositions And Methods For Allelic Gene Drive Systems And Lethal Mosaicism

Efficient super-Mendelian inheritance of transgenic insertional elements has been demonstrated in flies, mosquitoes, yeast, and mice. While numerous potentially impactful applications of such so-called gene-drive systems have been proposed they are currently limited to copying relatively large DNA cargo sequences (~1-10 Kb). Many desired genetic traits (e.g., drought tolerance in plants, crop yield, pest-resistance, or insecticide sensitivity), however, result from allelic variants altering only one or a few base pairs. An efficient system for super-Mendelian inheritance of such subtle genetic variants would accelerate a wide array of efforts to disseminate favorable traits throughout populations, or to assemble complex genotypes consisting of point-mutant alleles in combination with insertional transgenes for a multitude of research and applied purposes.

Applying a Machine Learning Algorithm to Canine Radiographs for Automated Detection of Left Atrial Enlargement

Researchers at the University of California, Davis have developed a method of detecting canine left atrial enlargement as an early sign of mitral valve disease by applying machine learning techniques to thoracic radiograph images.

Predictive Controller that Optimizes Energy and Water Used to Cool Livestock

Researchers at the University of California, Davis have developed a controller that applies environmental data to optimizing operations of livestock cooling equipment.

Single Conjugative Vector for Genome Editing by RNA-guided Transposition

The inventors have constructed conjugative plasmids for intra- and inter-species delivery and expression of RNA-guided CRISPR-Cas transposases for organism- and site-specific genome editing by targeted transposon insertion. This invention enables integration of large, customizable DNA segments (encoded within a transposon) into prokaryotic genomes at specific locations and with low rates of off-target integration.

A Wearable Platform for In-Situ Analysis of Hormones

UCLA researchers in the Department of Electrical and Computer Engineering have developed a highly sensitive, wearable hormone monitoring platform.

Improved Cas12a Proteins for Accurate and Efficient Genome Editing

Mutated versions of Cas12a that remove its non-specific ssDNA cleavage activity without affecting site-specific double-stranded DNA cutting activity. These mutant proteins, in which a short amino acid sequence is deleted or changed, provide improved genome editing tools that will avoid potential off-target editing due to random ssDNA nicking.

Method To Implement A Crispr-Cas9 Copycat Gene Drive In Rodents

Currently, alleles at multiple loci in the mouse genome must be combined by Mendelian genetics in crosses of animals to one another to produce a desired compound mutant genotype. For example, to combine homozygous mutations at two loci, animals that are heterozygous for each gene must be produced by breeding, and these are subsequently crossed to one another. Since the frequency of homozygosity for each allele is 1:4 the frequency of homozygosity for both genes is 1:16. Since the average litter of mice is approximately 10 pups, and the generation time from conception to reproductive age is about 3 months, this requires a substantial number of animals and time. With the addition of each new locus (three, four, etc), the cost measured in animals, time, and money increases exponentially. These factors increase substantially more if two or more loci are genetically linked, which requires rare recombination events to combine engineered alleles on the same chromosome. The CRISPR-Cas9 gene drive system stands to revolutionize rodent breeding. If each desired allele is encoded as a gene drive element that contains an sgRNA designed to target the same genomic location in the wild type homologous chromosome, each locus will be “driven” to homozygosity in the presence of Cas9. Therefore, in order to combine three alleles, for example, a mouse with one gene drive element (A) would be crossed to a mouse that encodes Cas9. Offspring of this cross would then be crossed to mice carrying gene drive element B, and these offspring would be crossed to mice carrying gene drive element C. In the presence of Cas9 at each generation, these gene drive elements at three distinct loci will be converted to homozygosity such that 50% of offspring, those that inherit Cas9, will be triple homozygous after three generations, even if they are genetically linked loci. A CRISPR-Cas9 mediated gene drive leverages the native cellular mechanism of homology directed repair to copy a desired allele from one chromosome to another. This process can convert a heterozygous genotype to homozygosity in a single generation. While CRISPR-Cas9 gene drives have been implemented in two species of insects, flies and mosquitos, it has not been reported in any non-insect animal species. 

DNA Methylation: A New Method for the Quantitative Predictor Of Age In Dogs

The ability to properly estimate the age of dogs would be quite useful in a variety of ways. For example, proper age estimation is important because age often plays a significant role when making medical decisions for pets. Currently, the accepted method to estimate age in dogs is based on the quality of teeth as well as ocular features. Estimating age based on tooth-wear (the commonly used metric in shelters) is very inaccurate after the teeth have fully erupted, generally by 6-7 months of age in dogs. Unfortunately, these methods have an accuracy of ~50% at best for domesticated pets and is error-prone for dogs between 2-8 years, encompassing a large portion of a dog’s adult life. Thus, shelters commonly underestimate the ages of these dogs to increase the likelihood of dogs being adopted, as people generally have a preference for younger pets. 

Targeted Ionophore-Based Metal Supplementation

Metal deficiency is implicated in a variety of genetic, neurological, cardiovascular, and metabolic diseases. Current approaches for addressing metal deficiency rely on generic metal ion supplementation, which can potentially lead to detrimental off-target metal accumulation in unwanted tissues and subsequently trigger oxidative stress and damage cascades. The inventors have developed a new modular platform for delivering metal ions in a tissue-specific manner and demonstrate liver-targeted copper supplementation as a proof of concept of this strategy. Specifically, the inventors designed and synthesized a N-acetylgalactosamine-functionalized ionophore, Gal-Cu(gtsm), to serve as a copper-carrying “Trojan Horse” that targets liver-localized asialoglycoprotein receptors (ASGPRs) and releases copper only after being taken up by cells, where the reducing intracellular environment triggers copper release from the ionophore. The inventors utilized a combination of bioluminescence imaging and inductively-coupled plasma mass spectrometry assays to establish ASGPR-dependent copper accumulation with this reagent in both liver cell culture and mouse models with minimal toxicity. The modular nature of this synthetic approach presages that this platform can be expanded to deliver a broader range of metals to specific cells, tissues, and organs in a more directed manner to treat metal deficiency in disease. This patent broadly covers directed metal delivery to select organs, tissues, and organelles.

Phenotypic Age And DNA Methylation Based Biomarkers For Life Expectancy And Morbidity

UCLA researchers in the Departments of Human Genetics and Biostatistics have combined phenotypic age and DNA methylation patterns to developed a novel biomarker for biological age.

Composition Of Matter And Method For Leptospirosis Vaccine

Leptospirosis is one of the most widespread diseases estimated to infect up to 7-10 million people per year worldwide (2014) that can be transmitted from animals to humans. The most common transmission is via the urine of rodents or domestic animals that contaminates water or soil. Unfortunately, it can cause severe infection and currently there is not an efficient vaccine present to combat this disease. The disease is caused by Leptospira, a genus of the spirochaete bacteria of which there are ~13 pathogenic species that effect humans. The signs and symptoms of the disease are quite variable and can range from mild headaches, muscle pains, and fevers to the more severe form which causes bleeding from the lungs.

Human Respiratory Disease Model Developed from Titi Monkey Adenovirus

Researchers at the University of California, Davis have cultured a titi monkey adenovirus (TMAdV,) and used the virus to develop a model of human respiratory disease.

Label Free Assessment Of Embryo Vitality

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

A Therapeutic Bandage For Treating Venom-Induced Tissue Necrosis

Snakebites affect 4.5 million people a year. Treatments that could save victim’s lives require capable medical facilities, which are not always within reach. Researchers at UCI have developed a therapeutic bandage for treating the tissue necrosis associated with snake bites. The invention can be applied in the field and has the potential to save many lives.

Enhanced Cell/Bead Encapsulation Via Acoustic Focusing

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 within the device.Counteracting these vortices allows for single particle encapsulation in the droplets formed by the device and thereby makes this technology a good candidate for use in single cell diagnostics and drug delivery systems.

A Micro/Nanobubble Oxygenated Solutions for Wound Healing and Tissue Preservation

Soft-tissue injuries and organ transplantation are common in modern combat scenarios. Organs and tissues harvested for transplantation need to be preserved during transport, which can be very difficult. Micro and nanobubbles (MNBs) offer a new technology that could supply oxygenation to such tissues prior to transplantation, thus affording better recovery and survival of patients. Described here is a novel device capable of producing MNB solutions that can be used to preserve viability and function of such organs/tissue. Additionally, these solutions may be used with negative pressure wound therapy to heal soft-tissue wounds.

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