Existing RNA-targeting tools for sequence-specific manipulation include anti-sense oligos (ASOs), designer PUF proteins and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas systems. However, there are significant limitations to each of the current tools. ASOs are usually not available for most RNA manipulations other than gene silencing. Designer proteins, such as PUF (Pumilio and FBF homology protein), possess low RNA recognition efficiency and it remains challenging to target RNA sequences >8-nucleotides (nt) in length. The bulky Cas protein (Cas13d: average 930 amino acids) leads to complication for transgene delivery and concerns of its immunogenicity due to its bacterial origin. Mutants of zinc finger(ZnF) proteins in ZRANB2 recognize a single-strand RNA containing a novel GGG motif with micromolar affinity, compared to the original motif GGU. These mutants serve as a foundation for RNA-binding ZnF designer protein engineering for in vivo RNA sequence-specific targeting.ZnFs are generally compact domains (~3kDa each) that have been successfully engineered for DNA recognition as modular arrays. A ZnF-based system has unique advantages, especially in a therapeutic context: (1) Broad application with the possibility to fuse with other effector domains; (2) High efficiency of RNA recognition (3 RNA bases recognized per 30-amino-acid ZnF) with a small size of protein. Only 4 ZnFs (~100 aa) is required for specific targeting in the transcriptome. (3) Humanized components without immunogenic concern.By engineering new sequence specificity of the ZRANB2 ZnF1, researchers from UC San Diego identified 13 mutants that altered their preferred RNA binding motif from GGU to GGG. They are N24R, N24H, N14D/N24R, N14D/N24H, N14R/N24R, N14R/N24H, N14H/N24R, N14H/N24H, N14Q/N24R, N14Q/N24H, N14E/N24R, N14S/N24R, N14E/N24H.

Reseachers from UC San Diego demonstrated a proof of principle for a CAGEX RNA-targeting CRISPR–Cas13d system as a potential allele-sensitive therapeutic approach for HD, a strategy with broad implications for the treatment of other neurodegenerative disorders.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a serious threat to human health without effective treatment. There is an urgent need for both real-time tracking and precise treatment of the SARS-CoV-2 infected cells to mitigate and ultimately prevent viral transmission. However, selective and responsive triggering and tracking of the therapeutic processin infected cells remains challenging.

Researchers from UC San Diego developed an invention that enables protein expression to be upregulated using specific proteins and/or peptide sequences derived from SARS-CoV-2 proteins that are engineered to recognize specific mRNA transcripts by fusion to RNA-targeting modules such as CRISPR/Cas systems. They anticipate that these proteins can be fused or tethered to any engineered RNA-targeting moiety/module such as PUF/Pum, and pentatricopeptide proteins.

UC San Diego researchers synthesized a cell‐penetrating NEMOActPep where the NEMO peptide was fused to a peptide known to penetrate cell membrane. They also synthesized the corresponding mutant version where all six critical amino acids within this NEMOActPep were mutated to glycines.Currently, UC San Diego is looking for a company interested in developing US Patent Rights.

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.

Generation of drug-like molecules with high binding affinity to target proteins remains a difficult and resource-intensive task in drug discovery. Existing approaches primarily employ reinforcement learning, Markov sampling, or deep generative models guided by Gaussian processes, which can be prohibitively slow when generating molecules with high binding affinity calculated by computationally-expensive physicsbased methods. Researchers a UC San Diego have developed a new approach, named Latent Inceptionism on Molecules (LIMO), which significantly accelerates molecule generation with an inceptionism-like technique. LIMO employs a variational autoencoder-generated latent space and property prediction by two neural networks in sequence to enable faster gradient-based reverse-optimization of molecular properties.

Considering the versatile functions of m6A in various physiological processes, it is thus not surprising to find links between m6A and numerous human diseases; many originated from mutations or single nucleotide polymorphisms (SNPs) of cognate factors of m6A. The linkages between m6A and numerous cancer types have been indicated in reports that include stomach cancer, prostate cancer, breast cancer, pancreatic cancer, kidney cancer, mesothelioma, sarcoma, and leukaemiaC-MYC(MYC) was among of the earliest described human oncogenes identified and is now recognized as the primary driver in oncogenic transformation and maintenance of cancer gene expression programs in a broad spectrum of cancer types where cells become “addicted” and dependent on MYC for survival. MYC transcript stability is coordinately regulated by RNA-binding proteins that both positively and negatively affect its half-life. Several RBPs interacting with m6A-modified RNA become upregulated in cancer, and are required for cellular growth, survival and invasion of cancer cells. Thus, Myc regulates cellular function and survival in part by modulating RNA metabolism and is itself controlled posttranscriptionally by RBPs.There have been conflicting findings regarding the function of YTHDF2 in cancer. For example, loss of YTHDF2 sensitizes acute myeloid leukemia (AML) cells to TNF-induced apoptosis, while overexpression of YTHDF2 in hepatocellular carcinoma (HCC) represses cell proliferation and growth by destabilizing EGFR mRNA [9, 10]. Moreover, the direct YTHDF2 target RNAs have yet to be defined in the mammary epithelial or in human breast cancer. It is unknown if the mechanism in other cancer types may be attributed to YTHDF2-Prss23 regulation. Existing art includes US20100104501A1 patent, characterizing Prss23 as a biomarker, therapeutic and diagnostic target. The invention involves compounds which bind to and/or inhibit the activity of PRSS23, which is the opposite of what we have determined is required to trigger apoptosis in Myc-dependent cancer.There are no current findings suggesting intervention of the YTHDF2-Prss23 binding interaction as a cancer therapeutic.

Cellular lipid membranes are embedded with transmembrane proteins crucial to cell function. Elucidating membrane proteins’ diverse structures and biophysical mechanisms is increasingly necessary due to their growing prevalence as a therapeutic target and sheer ubiquity in cells. Most biophysical characterization strategies of transmembrane proteins rely on the tedious overexpression and isolation of recombinant proteins and their reconstitution in model phospholipid bilayers.Unfortunately, membrane protein reconstitution depends on the use of denaturing and unnatural detergents that can interfere with protein structure and function. We have developed a detergent‐free method to reconstitute transmembrane proteins in model phospholipid vesicles and GUVs. Additionally, transmembrane proteins are difficult to express in cells due to the extreme insolubility of their transmembrane domain. By incorporating a synthetic transmembrane peptide into liposomes and simply expressing soluble portions of transmembrane proteins in cells, we can use this semisynthetic ligation strategy to more easily construct functional transmembrane proteins and reconstitute them into liposomes for biophysical and biochemical studies.Inteins can be found contiguously or non contiguously within some proteins. Non‐contiguous inteins are called “split inteins”. Inteins can be thought of as a type of protein intron which splices itself out of proteins. When non‐contiguous inteins find and bind to each other, they are then able to excise themselves resulting in the ligation of their respective exteins. Split intein pairs (C‐intein and N‐intein) can be attached to proteins of interest in synthetic and cellular systems to ligate protein sequences together.

Existing art in modulation of gene expression by nucleic acid targeting mechanisms primarily comprises methods for REDUCING gene expression, e.g. via DNA targeting (CRISPR gene knockout, reduction of transcription via CRISPR-i), or RNA targeting (shRNAs/siRNAs, ASOs, microRNA mimics). ENHANCEMENT of gene expression on the RNA level has been achieved using microRNA inhibitors; however the effects are typically small and are not target-specific (many other microRNA target-RNAs are also upregulated).The molecular functions of the majority of RNA-binding proteins (RBPs) remain unclear, highlighting a major bottleneck to a full understanding of gene expression regulation. 

While splice modulators have entered clinical trials, limited clinical efficacy in splicing factor mutation-driven malignancies, such as acute myeloid leukemia, has remained a challenge. There is a pressing unmet medical need for developing potent small molecule splice modulators for the treatment of a broad array of malignancies characterized by splicing deregulation.  However, the inability to practically access gram-scale lead molecules with viable pharmacological properties continues to hinder their application.

Bioorthogonal ligations encompass coupling chemistries that have considerable utility in living systems. Among the numerous bioorthogonal chemistries described to date, cycloaddition reactions between tetrazines and strained dienophiles are widely used in proteome, lipid, and glycan labeling due to their extremely rapid kinetics. In addition, a variety of functional groups can be released after the cycloaddition reaction, and drug delivery triggered by in vivo tetrazine ligation is in human phase I clinical trials. While applications of tetrazine ligations are growing in academia and industry, it has so far not been possible to control this chemistry to achieve the high degrees of spatial and temporal precision necessary for modifying mammalian cells with single-cell resolution.

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.

Resent strategies aimed to target and manipulate RNA in living cells mainly rely on the use of antisense oligonucleotides (ASO) or engineered RNA binding proteins (RBP). Although ASO therapies have been shown great promise in eliminating pathogenic transcripts or modulating RBP binding, they are synthetic in construction and thus cannot be encoded within DNA. This complicates potential gene therapy strategies, which would rely on regular administration of ASOs throughout the lifetime of the patient. Furthermore, they are incapable of modulating the genetic sequence of RNA. Although engineered RBPs such as PUF proteins can be designed to recognize target transcripts and fused to RNA modifying effectors to allow for specific recognition and manipulation, these constructs require extensive protein engineering for each target and may prove to be laborious and costly. 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-top:0in; mso-para-margin-right:0in; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0in; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; 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; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

Ulcerative colitis (UC), an inflammatory bowel disease (IBD), is characterized by chronic inflammation of the colon, with severity of mucosal inflammation being associated with a higher risk of work disability, hospitalization, colorectal cancer, and colectomy. Non-specific immunosuppressive agents targeting the host, such as steroids, thiopurines, and/or biologics, are used to offset the natural history of disease in patients with moderate-severe inflammation. These therapies are, however, associated with significant risks and often ineffective in adequately managing disease. Genomic technologies have identified associations between microbial dysbiosis, or temporal shifts in composition, and UC severity. While recent efforts extended profiling of microbiota in UC beyond genomics, it remains poorly understood if these shifts are causal or associative in nature, and which mechanisms govern pathogenic roles of the microbiome in UC.

Pancreatic cancer is an aggressive disease with limited treatment options and a high mortality rate. Pancreatic cancer is the 3rd leading cause of cancer death in the United States; despite some recent advances in systemic therapy, survival remains dismal in large part due to its profound drug resistance and its propensity for early metastasis. Typically, diagnosis of pancreatic cancer occurs only with advanced stages of the disease since there are currently no early markers for detection. Individuals with pancreatic cancer have a poor prognosis due to the late diagnosis, the extent of metastasis, and ineffective treatments. Survival rates are dismal and pancreatic cancer is not typically responsive to radiation and chemotherapy. An alternative approach for the treatment of pancreatic cancer as well as the design of a new class of therapeutics that can be used to treat this devastating disease is an immediate unmet medical need.

Rho-family small (~21kDa) GTPases are essential for regulation of numerous cellular functions. There are 20 members of the Rho family in mammals, of which four (Rac1, Rac2, Rac3, RhoG) belong to the Rac subfamily. Each Rac GTPase functions as a molecular switch by cycling between an active GTP-bound form and an inactive GDP-bound form. In addition to their normal cellular functions, Rac GTPases contribute to cancer development as downstream effectors of growth factor receptor signaling and oncogenic mutations in the Ras pathway. Rac GTPases represent attractive targets for therapy in hematologic cancer, however direct targeting of small GTPases has proved difficult and largely ineffective. A thorough understanding of the diverse mechanisms controlling Rac activation in cancer will therefore be essential towards identifying new therapeutic avenues and improving outcomes in patients One insight into the regulation/activation of the Rac GTPases involves examining Ras proteins and their signal transduction pathways since mutations that produce abnormally active Ras proteins are found in 30% of all human cancers. Moreover, after activation, RAS signaling is mediated through interaction with RAS-binding domains or through the domain RAS association (RA), transmitted to downstream effectors. Notably, many downstream effectors are oncogenes or tumor suppressor genes that are mutated or silenced in cancers independently of RAS. Ras proteins are involved in Ras association domain-containing protein 2 (RASSF2) and it has recently been shown that in Acute myeloid leukemia cells with low expression of RASSF2 are more resistant to pharmacological inhibition of Dedicator of cytokinesis protein 2 (DOCK2), a guanine nucleotide exchange factor (GEF). Acute myeloid leukemia cells with high expression of RASSF2 are sensitive to pharmacological inhibition of DOCK2.

Nonalcoholic fatty liver disease (NAFLD) is a condition in which excess fat is stored in the liver, though not caused by heavy alcohol use. NAFLD is one of the most common causes of liver disease in the United States. NAFLD it typically asymptomatic but when NAFLD advances, it can result in the development of NASH (Nonalcoholic steatohepatitis) where inflammation and fibrosis are widespread in the liver, resulting in nonalcoholic steatohepatitis and liver cirrhosis. Mechanisms of NAFLD progression are poorly understood. Experts estimate that about 20% of people with NAFLD have NASH. Between 30% and 40% of adults in the United States have NAFLD. About 3% to 12% of adults in the United States have NASH. There are no existing FDA‐approved therapies for nonalcoholic fatty liver disease (NAFLD). NAFLD it typically asymptomatic but it can progress to nonalcoholic steatohepatitis and liver cirrhosis. Mechanisms of NAFLD progression are poorly understood. There are many FDA‐approved therapies for type 2 diabetes, including metformin, insulin, sulfonylureas, Glp‐1 receptor agonists, Dpp‐4 inhibitors, and Sglt2 inhibitors. These drugs work through diverse mechanisms such as increasing insulin secretion (sulfonylureas, Glp‐1 receptor agonists, Dpp‐4 inhibitors), direct insulin replacement (insulin), reducing glucose production by the liver (metformin), and stimulating excretion of glucose into urine (Sglt2 inhibitors).

Fibroblast-like synoviocytes (FLS) in the intimal lining of the joint synovium control the composition of the synovial fluid and extracellular matrix (ECM) of the joint lining. In rheumatoid arthritis (RA), FLS become aggressive and invasive, contributing to many aspects of RA pathology. FLS produce matrix metalloproteinases (MMPs) that break down the ECM, directly invade and digest the articular cartilage, promote bone erosion, and promote inflammation through secretion of interleukin 6 (IL-6), chemokines, and other inflammatory mediators. FLS are highly sensitive to the inflammatory environment present in rheumatoid joints. Growth factors, especially platelet-derived growvth factor (PDGF), stimulate FLS invasiveness. Inflammatory cytokines, particularly tumor necrosis factor-alpha (TNF) and interleukin-I (IL-1), enhance FLS aggressiveness, pro-inflammatory features and MMP production. Targeting of molecules that control FLS invasiveness and inflammatory output is being considered an option for development of new therapies for RA.   Many signaling pathways controlling FLS behavior rely upon phosphorylation of proteins on tyrosine residues, which results from the balanced action of protein tyrosine kinases (PTKs) and phosphatases (PTPs). We found that a protein (PTPRA) belonging to a novel and currently untapped class of drug targets is present at high levels in cells lining the joints of RA patients, where we believe it promotes the aggressive behavior of these cells in joint inflammation and destruction.

Nonalcoholic steatohepatitis (NASH), characterized by hepatic steatosis with inflammation and liver damage, has become the leading cause of transplant and liver associated death. Moreover, numerous studies suggest that hepatocellular death is the key event triggering progression to fibrosis and cirrhosis for NASH and perhaps other liver diseases.  In normal liver, hepatocyte apoptosis plays a key role in liver homeostasis, maintaining equilibrium between the loss and replacement of hepatocytes. However, pathological conditions such as viral infection, alcoholic or nonalcoholic steatohepatitis and physical injury, lead to extensive hepatocyte apoptosis and liver damage. While inflammation contributes to the pericellular fibrosis at an early stage, sustained liver damage leads to scarring, bridging fibrosis and subsequent development of cirrhosis. Moreover, hepatocellular death is the major contributor to the pathogenesis of cirrhosis and hepatocellular carcinoma. Therefore, understanding the molecular mechanisms by which hepatocellular death is controlled may lead to new treatments for liver diseases.

Pathological accumulation of phosphorylated Tau (pTau) and accumulation of amyloid-beta (Ab) fragments are the two major biochemical hallmarks of Alzheimer’s disease (AD). Effective strategies to remove Ab in AD-patient brains have been developed, but have not yet shown efficacy to slow cognitive decline in clinical trials. This finding has led to the idea that targeting Tau or combinatorial strategies that target both Tau and Ab are required to treat AD. Genetic, epidemiologic, and biochemical evidence suggests that predisposition to AD may arise from altered cholesterol metabolism, although the molecular pathways that may link cholesterol to AD phenotypes are only partially understood. Stimulation of a brain specific cytochrome that converts cholesterol to 24-hydroxycholesterol, which in turn reduces cholesteryl ester. Reduction of cholesteryl ester has been demonstrated to reduce pathological Tau phosphorylation in human neurons made from induced pluripotent stem cells. Also, low dose Efavirenz/Sustiva reduces neurofibrillary tangles in a mouse model. The pathway may run from cholesteryl ester to Tau via the proteasome.

Human Ubiquitin-like modifier-activating enzyme 7 (UBA7) is a protein is involved in protein modification, specifically involving the pathway for protein ubiquitination. The modification of proteins with ubiquitin is an important cellular mechanism for targeting abnormal or short-lived proteins for degradation. Ubiquitination involves at least three classes of enzymes: ubiquitin-activating enzymes, or E1s, ubiquitin-conjugating enzymes, or E2s, and ubiquitin-protein ligases, or E3s. UBA7  encodes a member of the E1 ubiquitin-activating enzyme family. Moreover, ubiquitination and ubiquitin-like post-translational modifications (PTMs) regulate activity and stability of oncoproteins and tumor suppressors. Biomarkers are very important as companion diagnostic tools to guide clinical practice in treating human cancers, especially for targeted therapies. In the era of precision medicine, it is important for development companion diagnostic tools that can guide clinical practice for treating human cancers using targeted therapies.

Human immunodeficiency virus type-1 (HIV-1) is a pathogenic retrovirus and the causative agent of acquired immunodeficiency syndrome (AIDS) and AIDS-related disorders. There were 1.7 million new infections globally in 2018, and ~38 million people are currently living with HIV-1. Although the introduction of antiretroviral therapy (ART) has prevented millions of AIDS-related deaths worldwide, patients must continue to receive ART for the remainder of their lives. HIV-1 reservoirs persist even while subjects are on ART, leading to a rapid increase in viral replication when therapy is discontinued. Therefore, eradication of persistent HIV-1 reservoirs remains the main barrier to achieving a cure for HIV-1/AIDS. The prevailing view of persistence suggests that the virus remains in a latent state in memory CD4+ T cells regardless of plasma viral loads, allowing the virus to establish a life-long infection in the host. Since the latent virus is refractory to existing antiretroviral therapies, curative strategies are now focusing on agents that reactivate viral replication and render it susceptible to conventional therapy. Any strategy aimed at controlling and eradicating viral reservoirs in HIV-1-infected individuals must target such latent reservoirs. The mammalian genome encodes thousands of long noncoding RNAs (lncRNAs, >200 nucleotides), including intergenic lncRNAs (lincRNAs), which are increasingly recognized to play major roles in gene regulation. The pathophysiological functions and mechanisms of lncRNAs in gene regulation have started to emerge. Work over the last few years has begun to uncover the role of lncRNAs in modulating HIV-1 gene expression.

The existing CAR-engineered T cell-based (CAR-T) therapy represents one of the most successful immunotherapy approaches developed in recent years. Most CAR-T cell therapy has been used clinically to treat hematological malignancies by targeting the B cell-specific antigen, CD19. However, this approach is not without limitations due to toxicities such as by neurotoxicity or cytokine release syndrome. Additionally, CAR-T cells function only as autologous cells due to graft-versus-host disease that would develop if cells were obtained from another person. Therefore, CAR-T cells must be produced on a patient-specific basis. NK cells, on the other hand, function as allogenic cytotoxic effector cells that do not have to be utilized on a patient-specific basis and are proven to be less toxic since they do not cause cytokine release syndrome, neurotoxicity, or graft-versus-host disease. For these reasons, CAR-engineered NK (CAR-NK) cells have increasingly attracted interest as an alternative CAR-cell therapy. However, there exist other unmet challenges. Targeting CAR-based therapies against solid tumors has been challenging due to the lack of truly tumor-specific antigens as most targets are shared by non-malignant cells and can cause toxicity due to “on-target, off-tumor” effects.” A fine-tunable CAR therapy is useful to better identify and target tumors while limiting this toxicity.

Schistosomiasis is a disease caused by infection with parasitic flatworms called schistosomes. The three major medically important species are Schistosoma mansoni (causing intestinal schistosomiasis in Africa and South America), S. japonicum (intestinal schistosomiasis in East Asia), and S. haematobium (causing genitourinary schistosomiasis in Africa and the Middle East). Signs and symptoms may include abdominal pain, diarrhea, bloody stool, or blood in the urine.  The treatment of schistosomiasis serves three purposes: reversing acute or early chronic disease, preventing complications associated with chronic infection, and preventing neuroschistosomiasis. The goal of treatment is to remove the worms that produce the eggs which, in turn, are responsible for disease morbidity and mortality. There is no effective vaccine against schistosomiasis.