Niren Murthy, Ph.D. - US grants

0546962
Murthy
The applicant proposes to use the T1/T2 quenching effect for imaging protease activity, and he has demonstrated in preliminary results a combined T2 and T1 agent (Dysprosium and Gadolinium connected by a PEG chain) which has very small modulation to the MR signal intensity, due to the offsetting effect of the T2 and T1 agents. By design, the T2 agent is attached to the PEG chain through a trypsin cleavage site, which can be broken by a target enzyme (e.g. trypsin). Once detached from the PEG chain, the T2 agent loses its modulation effect on the MR signal, resulting in an intensity increase reflecting the protease activity. The applicant ultimately seeks to apply this methodology to image the activity of MMP-7 in mouse tumors, since it is over-expressed in several cancers.

[unreadable] DESCRIPTION (provided by applicant): Superoxide dismutase (SOD) is an enzyme that can inhibit the production of ROS and has shown tremendous promise for the treatment of acute liver failure in animal models. Unfortunately, SOD has performed poorly in clinical trials because of drug delivery problems. The objective of this R21 application is to develop a new class of polymeric nanoparticles that can deliver SOD in vivo and treat acute liver failure. This new family of nanoparticles are termed the polyketal nanoparticles (PKNs). The central hypothesis of this proposal is that the: The PKNs have the physical and chemical properties needed to deliver SOD to Kupffer cells in vivo, inhibit the production of ROS, and treat acute liver failure. This hypothesis is based upon the unique chemistry of the PKNs and our preliminary findings, which indicate that the PKNs degrade rapidly (1-2 days) under the acidic conditions of the endosomes and lysosomes, target Kupffer cells in vivo, and do not generate acidic degradation products after hydrolysis. The experiments in this proposal will test our central hypothesis; by determining the ability of SOD encapsulated in the PKNs to inhibit ROS generation in liver macrophages and protect mice from Tylenol induced acute liver failure. The successful completion of this R21 application will demonstrate that the PKNs can deliver proteins to macrophages in vivo and will generate a potential treatment for acute liver failure. Furthermore, the PKNs have the potential to deliver small organic molecules, DNA and protein therapeutics. Given the wide range of diseases that macrophages and phagocytic cells are involved in, we anticipate that the PKNs will find widespread use in the field of drug delivery. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Atherosclerosis and atherosclerosis related complications such as heart attack, stroke, and angina, are the leading cause of death in the western world, and effective treatments are greatly needed. A key issue limiting the treatment of atherosclerosis is an inability to diagnose atherosclerotic plaques that have a high risk of growing and rupturing, also known as active plaques. The overproduction of reactive oxygen species (ROS) is necessary for the growth and rupture of atherosclerotic plaques, making ROS an excellent diagnostic marker for active plaques. The objective of this application is to develop a new family of fluorescent contrast agents, termed the hydrocyanines, which have the physical and chemical properties needed to image ROS in atherosclerotic plaques, in the carotid artery, and detect plaque activity. The hydrocyanines are a new family of fluorescent dyes, recently developed in our laboratory, which can detect ROS at nanomolar concentrations, have tunable emission wavelengths ranging from 570-810 nm, and accumulate within cells after oxidation. Our approach is to image ROS in the carotid artery of mice and rabbits, suffering from vascular injury and atherosclerosis, with the hydrocyanines. The central hypothesis of this proposal is that: The hydrocyanines have the physical and chemical properties needed to detect oxidative stress in the carotid artery and detect plaque activity. This hypothesis is based on our preliminary data, which demonstrates that the hydrocyanines can detect superoxide and the hydroxide radical in cell culture, in tissue samples and for the first time in vivo. The overall objective of this proposal will be accomplished by testing our central hypothesis through the following three Specific Aims; Specific Aim I: Optimize the hydrocyanines using the kinetic isotope effect Specific Aim II: Detect ROS production in a murine model of carotid artery injury Specific Aim III: Detect ROS production by atherosclerotic tissues in vivo in a rabbit model of atherosclerosis and ex-vivo in diseased human coronary arteries The experiments in this proposal will determine if the hydrocyanines can image ROS in atherosclerotic plaques and detect plaque activity in mice and rabbits. The experiments in this proposal are innovative because they will lead to the development of a contrast agent that can image ROS, in vivo, for the first time. This proposal is also significant because it will generate a new class of diagnostics, which have the potential to diagnose patients at risk of developing heart attacks and strokes. Furthermore, numerous other diseases are also associated with an overproduction of ROS, such as cancer and neurodegenerative disease; we therefore anticipate that the results of this proposal will impact several areas of medicine and biology.

DESCRIPTION (provided by applicant): Drug resistant Gram-negative bacteria are a major cause of mortality and morbidity in the world and new strategies for treating them are greatly needed. The objective of this proposal is to develop a new strategy for targeting ciprofloxacin into drug-resistant Gram-negative bacteria, composed of conjugating maltohexaose to antibiotics. Maltohexaose-conjugated antibiotics are designed to be selectively imported by bacteria via the maltodextrin (MD) transporter, but should be minimally internalized by mammalian cells due to their lack of MD transporters. After internalization, maltohexaose conjugated antibiotics are designed to be cleaved and release the free antibiotic within the bacterial cytoplasm. The central hypothesis of this proposal is that conjugates of maltohexaose with ciprofloxacin, termed MDC-1 and MDC-2, will be transported into drug-resistant P. aeruginosa by a factor of 10 greater than free ciprofloxacin, and will have increased antibiotic potency relative to that of free ciprofloxacin. In addition, we hypothesize that MDC-1 and MDC-2 will have a wider therapeutic window than free ciprofloxacin at treating drug resistant P. aeruginosa in a lung infection model. The overall objective of this proposal will be accomplished by testing our central hypothesis through the following Specific Aims: R21, Phase Specific Aim 1. Synthesis and intracellular transport of MDC-1 and MDC-2. Specific Aim 2. Antibacterial activity of MDC-1 and MDC-2 against drug resistant P. aeruginosa. The completion of the R21 phase experiments will demonstrate that the therapeutic efficacy of ciprofloxacin can be improved by conjugating it to maltodextrins. These studies will serve as the foundation for the subsequent R33 phase, which focuses on investigating the in vivo therapeutic efficacy and generality of MDC-1 and MDC-2 in fighting bacterial infections. R33 Phase, Specific Aim 1. Determine the in vivo efficacy of MDC-1 and MDC-2. Specific Aim 2. Determine the efficacy of MDC-1 and MDC-2 against Gram-negative bacteria. The experiments in this proposal are innovative because they will for the first time develop a small molecule strategy for targeting drugs to bacteria, and represent the first attempt to exploit the maltodextrin transport pathway for drug delivery. The proposed project is significant because it has the potential to generate a universal platform for enhancing the delivery of structurally diverse antibiotics into multidrug resistant bacteria. This new approach therefore has the potential to significantly impact medicine, public health and national security by reducing the emergence of antimicrobial-resistant organisms and stimulating the development of novel antibiotics.

? DESCRIPTION (provided by applicant): Bacterial infections of implanted medical devices are a growing problem in health care today and have a significant impact on morbidity, mortality and health care costs. For example, approximately 1 million patients acquire a nosocomial implant infection in the US every year, and the financial impact of these infections is in the billions of dollars annually. A major challenge in treating bacterial implant infections is diagnosing them at an early stage. At present, the diagnosis of an infection associated with a medical device is made on the basis of clinical criteria, such as physical exam and microbial cultures, which is problematic because this methodology can only detect late stage infections that are challenging to treat. Medical device infections are therefore frequently undiagnosed and allowed to progress to an advanced stage, resulting in significant morbidity and mortality. Conversely, medical devices with sterile inflammation may be inappropriately removed, based on clinical judgment alone, incurring increased morbidity and healthcare costs. In this clinical environment, a technology that can specifically detect infected implanted devices at an early stage, and distinguish them from sterile inflammation has the potential to have a significant clinical and economic impact. The central objective of this proposal is to develop and rigorously evaluate a new family of PET imaging contrast agents that can image implant infections by targeting the maltodextrin transporter. We have termed these agents the 18F-MDPs as they are composed of 18F conjugated to maltodextrins. 18F-MDPs have high specificity and sensitivity for bacteria because they are internalized by bacteria as a food source via the maltodextrin transport pathway, but are not internalized by mammalian cells as mammalian cells lack maltodextrin transporters. The experiments in this proposal focus on developing a novel conjugate of maltodextrin for use in PET imaging of bacteria and determining its efficacy in imaging infected medical implants. The central hypothesis of this proposal is that: Conjugates of maltodextrins with 18F will detect bacterial infections of implanted medical devices in a highly specific and sensitive manner. The following Specific Aims are proposed: Specific Aim 1: Synthesis and metabolism of 18F-labeled maltohexaose, 18F-thiomaltose and 18F- deoxythiomaltose Specific Aim 2: Internalization and metabolism of 18F-labeled maltohexaose, 18F-thiomaltose and 18F-deoxythiomaltose by bacteria known to cause medical device infections Specific Aim 3: Comparative in vivo imaging of medical device infections in rats with 18F-MDPs

? DESCRIPTION (provided by applicant): This project called Consortium for drug-resistant Gram-negative pathogen detection is an academic-industrial partnership established to address the challenges posed by the RFA Partnerships for Diagnostics to Address Antimicrobial Resistance of Select Bacterial Pathogens. The project will focus on two groups of Gram-negative bacterial (GNB) pathogens-carbapenem-resistant Enterobacteriaceae (CRE) and drug-resistant Pseudomonas aeruginosa (PA), which have been designated by the Centers for Disease Control and Prevention (CDC) as urgent threat and serious threat pathogens, respectively. These organisms are major causes of healthcare-associated infections (HAIs), but CREs are also emerging as life-threatening causes of community-onset infections (COIs). They are enormously complicated to treat, especially when they express enzymes (ß-lactamases) that inactivate commonly-used antimicrobial agents. This project will develop and validate an integrated diagnostic system designed to directly detect and differentiate GNBs into CREs and PA from blood and urine samples. The same system will be designed to detect all of the clinically important ß- lactamases, so that clinicians can make a more informed and rapid decision about which antibiotics to initiate. The integrated system is thus designed to mimic in one device most of the steps involved in a clinical microbiology laboratory to detect CREs and PA. The proposed diagnostic system is made possible because of two important innovations. One is an integrated molecular diagnostics system (iMDx) designed to rapidly separate GNBs from clinical sample and capture target GNBs for species identification. The other is called self-accelerated dimerization (SAD) assay that facilitates bacterial enzyme amplification so that the target enzyme activity can be detected in less than 10 minutes. The SAD assay will be integrated into iMDx. Together, these innovations will be refined and validated against a large panel of clinical isolates of GNBs obtained from blood stream (BSI) and urinary tract infections (UTI) in different regions of the world. The accuracy of the system will also be prospectively assessed at San Francisco General Hospital among patients hospitalized for BSI or sepsis, and catheter-associated UTI. A successful development of this system will not only improve patient clinical management, but help reduce unnecessary use of expensive, later-generation antibiotics and prevent the selection of new drug-resistant GNB pathogens in healthcare and community settings.

? DESCRIPTION (provided by applicant): The development of antibiotics against drug resistant gram negative bacteria (GNB) is a central problem in medicine. Developing antibiotics against GNB has been challenging because of their low membrane permeability, which prevents antibiotics from reaching therapeutic concentrations within GNB. The objective of this proposal is to develop a new strategy for enhancing the transport of antibiotics into GNB, via conjugation to thiomaltose. Thiomaltose conjugated antibiotics have internalization rates into GNB that are dramatically enhanced over free antibiotics because they enter bacteria via maltodextrin and other oligosaccharide transporters, which are the preferred food source for bacteria. In addition, mammalian cells do not express maltodextrin and oligosaccharide transporters, and therefore thiomaltose conjugated antibiotics should not enter mammalian cells and should have a much higher therapeutic window than free antibiotics. The experiments in this proposal will investigate if the efficacy of the drug radezolid can be improved by conjugating it to thiomaltose. The central hypothesis of this proposal is that: Radezolid conjugated to thiomaltose, termed TMR, will have a wider therapeutic window than free radezolid at treating drug resistant GNB, due to its enhanced transport and lower toxicity. This hypothesis is based on our preliminary data, which demonstrates that thiomaltose-conjugated radezolid is at least 2 orders of magnitude more effective at treating drug resistant P. aeruginosa than free radezolid. We will test the central hypothesis of this proposal by completing the following Specific Aims: R21 Phase Specific Aim 1. Increase the transport of radezolid into drug resistant GNB via conjugation to thiomaltose Specific Aim 2. Increase the efficacy of radezolid via conjugation to thiomaltose R33 Phase Specific Aim 1. Increase the in vivo efficacy of radezolid against GNB via conjugation to thiomaltose Specific Aim 2. Pharmacokinetics of TMR and optimization of its oral bioavailability The experiments in this proposal are innovative because they develop a strategy, which can target antibiotics to GNB and increase their therapeutic window. The experiments in this proposal are significant because they will lead to the development of new therapeutics for drug resistant GNB.

Abstract Duchenne muscular dystrophy (DMD) is a genetic disease caused by mutations in the dystrophin gene and causes thousands of deaths each year. There are no effective treatments for DMD and new DMD therapeutics are urgently needed. Cas9 based therapeutics have the potential to revolutionize the treatment of DMD, because they can correct dystrophin mutations, via homology directed DNA repair. However, developing Cas9 based therapeutics for DMD has been challenging because it requires simultaneously delivering Cas9 protein, guide RNA, and donor DNA in vivo and delivery vehicles have not been developed that can accomplish this. The central objective of this proposal is to develop a new family of nanoparticle delivery vehicles, termed CRISPR-Nanoparticles, which are designed to treat DMD by delivering Cas9 protein, guide RNA and donor DNA in vivo. CRISPR-Gold is our first generation CRISPR-Nanoparticle and is composed of gold nanoparticles complexed with Cas9 RNP, donor DNA and the endosomal disruptive polymer PASp(DET). We have been able to demonstrate that CRISPR-Gold, can correct 5% of the dystrophin mutations (via HDR) in muscle fibers after a direct injection in mdx mice, and thus has tremendous potential as a treatment for DMD. A successful DMD therapeutic needs to be able to correct 20% of the dystrophin mutations in muscle tissue, and the experiments in this proposal focus on developing 2nd and 3rd generation CRISPR-Nanoparticles, which can generate a 20% HDR rate in muscle tissue. In particular, the 2nd and 3rd generation CRISPR- Nanoparticles address the key factors preventing CRISPR-Gold from generating a 20% HDR rate in a clinical setting, which are (1) its lack of biodegradability, (2) its toxicity, and (3) the lack of cell division in muscle tissue. The central hypothesis of this proposal is that: Delivery vehicles that complex Cas9 protein, guide RNA, donor DNA and endosomal disruptive polymers will be able to efficiently induce HDR and treat DMD. The central objective of this proposal will be accomplished by completing the following Specific Aims. Specific Aim 1: Develop biodegradable CRISPR-Nanoparticles that can correct dystrophin mutations Specific Aim 2: Develop biocompatible CRISPR-Nanoparticles that can correct dystrophin mutations Specific Aim 3: Enhance the HDR efficiency of CRISPR-Nanoparticles with FDA approved stimulating agents At the conclusion of this proposal we will have identified a CRISPR-Nanoparticle formulation that can efficiently correct dystrophin mutations in vivo and has the biocompatibility needed for clinical translation. The experiments in this proposal are innovative because CRISPR-Gold is the first example of a delivery vehicle that can simultaneously deliver RNA, DNA and protein in vivo and induce HDR. The experiments in this proposal are significant because they will lead to the development of a new therapeutic for DMD (and other genetic diseases) that has the HDR efficiency and biocompatibility needed to enter into clinical trials.

CRISPR-based gene editing of the brain has the potential to revolutionize the treatment of neurological diseases. A large number of incurable brain diseases, such as Huntington's, Alzheimer's and Parkinson's disease, are caused by the over-expression of pathogenic proteins and could be treated with CRISPR based therapeutics. However, despite its potential, developing CRISPR based therapeutics for the brain has been challenging because of delivery problems. In particular, two key challenges need to be solved before gene editing in the brains of large animals and in humans is possible. First, strategies for efficiently and safely delivering Cas9 and gRNA into neurons, after an intracranial injection, need to be developed. Second, strategies that can enable a large volume of brain tissue (> 1 cm) to be transfected after an intracranial injection of CRISPR reagents also need to be developed. The central objective of this proposal is to develop a delivery strategy for gene editing the brains of large animals after an intracranial injection, termed convection-enhanced CRISPR (C-CRISPR). C-CRISPR is based on using convection-enhanced delivery (CED) to deliver an engineered Cas9 RNP, which has been fused to multiple nuclear localization signals (NLS), and has been encapsulated in PEGylated block copolymers. C-CRISPR addresses the key translational bottlenecks that have prevented CRISPR from having a translational impact in the brain. In particular, because it delivers the Cas9 RNP directly, it avoids the toxicity problems of viruses and the manufacturing challenges of using mRNA, and consequently has great translational potential. In addition, C-CRISPR uses CED to distribute the Cas9 RNP across centimeters of brain tissue, and therefore has the potential to edit the brains of large animals. C-CRISPR is based on our preliminary data demonstrating that the Cas9 RNP fused to multiple NLS signals can edit genes in murine brains after an intracranial injection, and that Cas9 RNP complexed to PEG-block copolymers can be delivered to centimeters of brain tissue, in the striatum, after delivery via CED. CED of engineered Cas9 RNP complexed to PEG block copolymers, therefore, has the potential to edit genes in human patients. We propose therefore the following aims/milestones: UG3 Specific Aim 1. Develop C-CRISPR formulations that distribute throughout the striatum of rats UG3 Specific Aim 2. Develop C-CRISPR formulations that edit centimeters of brain tissue UH3 Specific Aim 1. Develop C-CRISPR formulations that edit centimeters of tissue in pig brains The experiments in this proposal are significant because, if successful, C-CRISPR will be the first example of a non-viral delivery strategy that can edit genes in the brains of large animals. The experiments in this proposal are innovative because C-CRISPR is the first example of a delivery strategy that effectively integrates 3 complementary technologies, (1) engineered Cas9 RNPs (2) PEGylation and (3) convective enhanced diffusion, and will provide a roadmap for developing strategies for gene editing in higher animals.

Bacterial infections ranging from heart valve infections (endocarditis) to bone infections (osteomyelitis) are a growing problem in health care today and cause significant morbidity and mortality. The unmet clinical need is our inability to diagnose bacterial infections at an early stage in a specific and sensitive manner. At present, heart valve infections, bone infections and abscesses are diagnosed on the basis of clinical judgment augmented by several non-specific imaging techniques (e.g., CT, MRI, white blood cell scans) and cultures. . However, these approaches are either indirect in the case of imaging or take several days in the case of cultures. The central objective of this proposal is to develop the next generation of maltodextrin based PET contrast agents that can image and diagnose early stage gram positive and gram negative infections. We have recently demonstrated that a new optimized maltodextrin targeting ligand termed thiomalto-triose that can image gram positive bacterial infections in vivo. The following specific aims are proposed: Specific Aim 1: Synthesis and Characterization of 18F thiomalto-triose based bacterial targeting ligands Specific Aim 2: Evaluation of lead compounds for the detection of gram negative infections in an animal model of bacterial osteomyelitis. Specific Aim 3: Evaluation of lead compounds for the detection of gram positive infections in an animal model of bacterial endocarditis

CRISPR-based gene editing of the brain has the potential to revolutionize the treatment of neurological diseases. A large number of incurable brain diseases, such as Huntington's, Alzheimer's and Parkinson's disease, are caused by the over-expression of pathogenic proteins and could be treated with CRISPR based therapeutics. However, despite its potential, developing CRISPR based therapeutics for the brain has been challenging because of delivery problems. In particular, two key challenges need to be solved before gene editing in the brains of large animals and in humans is possible. First, strategies for efficiently and safely delivering Cas9 and gRNA into neurons, after an intracranial injection, need to be developed. Second, strategies that can enable a large volume of brain tissue (> 1 cm) to be transfected after an intracranial injection of CRISPR reagents also need to be developed. The central objective of this proposal is to develop a delivery strategy for gene editing the brains of large animals after an intracranial injection, termed convection-enhanced CRISPR (C-CRISPR). C-CRISPR is based on using convection-enhanced delivery (CED) to deliver an engineered Cas9 RNP, which has been fused to multiple nuclear localization signals (NLS), and has been encapsulated in PEGylated block copolymers. C-CRISPR addresses the key translational bottlenecks that have prevented CRISPR from having a translational impact in the brain. In particular, because it delivers the Cas9 RNP directly, it avoids the toxicity problems of viruses and the manufacturing challenges of using mRNA, and consequently has great translational potential. In addition, C-CRISPR uses CED to distribute the Cas9 RNP across centimeters of brain tissue, and therefore has the potential to edit the brains of large animals. C-CRISPR is based on our preliminary data demonstrating that the Cas9 RNP fused to multiple NLS signals can edit genes in murine brains after an intracranial injection, and that Cas9 RNP complexed to PEG-block copolymers can be delivered to centimeters of brain tissue, in the striatum, after delivery via CED. CED of engineered Cas9 RNP complexed to PEG block copolymers, therefore, has the potential to edit genes in human patients. We propose therefore the following aims/milestones: UG3 Specific Aim 1. Develop C-CRISPR formulations that distribute throughout the striatum of rats UG3 Specific Aim 2. Develop C-CRISPR formulations that edit centimeters of brain tissue UH3 Specific Aim 1. Develop C-CRISPR formulations that edit centimeters of tissue in pig brains The experiments in this proposal are significant because, if successful, C-CRISPR will be the first example of a non-viral delivery strategy that can edit genes in the brains of large animals. The experiments in this proposal are innovative because C-CRISPR is the first example of a delivery strategy that effectively integrates 3 complementary technologies, (1) engineered Cas9 RNPs (2) PEGylation and (3) convective enhanced diffusion, and will provide a roadmap for developing strategies for gene editing in higher animals.