ADVANCED GENE TRANSFER COMPANY, INC.

2023-08-05

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Department of Health and Human Services

ENHANCED CRISPR GENE EDITING IN PLURIPOTENT STEM CELLS USING CARBON NANOTUBE ARRAYS - ABSTRACT THE GOAL OF THE PROPOSED STUDIES IS TO IMPROVE THE EFFICIENCY OF CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)-BASED GENE EDITING IN HUMAN PLURIPOTENT STEM CELLS (PSCS), INCLUDING EMBRYONIC STEM CELLS (ESCS) AND INDUCED PLURIPOTENT STEM CELLS (IPSCS). CURRENTLY, CRISPR/CAS9 SHOWS GREAT POTENTIAL FOR TARGETED GENE EDITING, BUT REMAINS CHALLENGING IN PSCS DUE TO THEIR BEING HIGHLY REFRACTORY TO CONVENTIONAL TRANSFECTION METHODS COMPARED TO OTHER PRIMARY CELL TYPES AND CELL LINES. THIS POSES A SIGNIFICANT HURDLE TO THE TARGETED GENETIC MANIPULATION OF PSCS. WHAT IS NEEDED IS A TRANSFECTION METHOD THAT IS FAST, EFFICIENT, REQUIRES FEWER INPUT CELLS, AND CAN INTRODUCE DNA/RNA/PROTEIN COMPLEXES INTO PSCS WITH MINIMAL TOXICITY. AGTC HAS DEVELOPED A NOVEL METHOD TO EFFICIENTLY INTRODUCE BIOMOLECULES INTO MAMMALIAN CELLS USING DEVICES COMPOSED OF AN ARRAY OF CLOSELY PACKED AND ALIGNED CARBON NANOTUBES (CNT) TO ACHIEVE HIGHLY EFFICIENT TRANSFER WITH LOW CYTOTOXICITY. AGTC HAS ALSO DEVELOPED A SCALABLE NANOMANUFACTURING PROCESS FOR THESE CNT DEVICES USING TEMPLATE-BASED CHEMICAL VAPOR DEPOSITION (CVD) TO PRODUCE A DEVICE CONSISTING OF THOUSANDS OF 200 NM- DIAMETER HOLLOW CARBON NANOTUBES (CNT) EMBEDDED IN A 13 MM-DIAMETER BASE WHICH CAN BE USED WITH STANDARD TISSUE CULTURE PLATES. IN THIS PROPOSAL, AGTC WILL USE CNT ARRAYS TO INCREASE THE EFFICIENCY OF TRANSFER OF PROTEIN AND NUCLEIC ACIDS INTO PSCS. THE HYPOTHESIS IS THAT THE UNIQUE GEOMETRY OF THE CNT DEVICE SURFACE IS CRITICAL TO BOTH CELL VIABILITY AND BIOMOLECULE TRANSFER, AND THAT CNT DEVICES WILL EFFICIENTLY TRANSFER DNA AND PROTEIN INTO PSCS. THE SPECIFIC AIMS OF THIS PHASE I PROPOSAL ARE: (1) ENHANCED PRODUCTION OF INDEL MUTATIONS IN HUMAN PSCS USING CRISPR DELIVERED BY CNT, AND (2) DEVELOP CNT-ENHANCED, HOMOLOGY-DIRECTED RECOMBINATION (HDR) IN PSCS. WE WILL TRANSFER PREPACKAGED RECOMBINANT CAS9 WITH GRNA AND HDR OLIGONUCLEOTIDES INTO IPSCS. FOR THESE STUDIES, WE WILL USE IPSCS THAT CONTAIN AN ENDOGENOUS EGFP GENE, AND MONITOR EDITING EFFICIENCY BY FLUORESCENCE ACTIVATED CELL SORTING (FACS), FLUORESCENCE MICROSCOPY, AND DNA SEQUENCING OF THE EGFP ALLELE. THESE STUDIES WILL ESTABLISH THE CONDITIONS AND EFFICIENCY FOR CRISPR GENE EDITING IN PSCS USING CNT ARRAYS FOR EFFICIENT DELIVERY OF NUCLEIC ACID/PROTEIN COMPLEXES. FUTURE PHASE 2 STUDIES WILL EXPAND THIS TO DEVELOP DEVICE FORMATS SUITABLE FOR EFFICIENT GENOME-WIDE CRISPR SCREENS AND RAPID GENERATION OF SYNGENEIC IPSCS FOR DISEASE MODELING.

275045.00

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2023-03-16

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Department of Health and Human Services

CARBON NANOTUBE-MEDIATED GENE TRANSFER INTO HUMAN T-CELLS FOR CAR-T HIV THERAPY - ABSTRACT THE HIV PANDEMIC HAS CAUSED AN ESTIMATED 33 MILLION DEATHS TO DATE. IN THE U.S. ALONE, 1.2 MILLION PEOPLE CURRENTLY LIVE WITH HIV AND 34,800 NEW CASES WERE DIAGNOSED IN 2019. THIS ADDS UP TO AN ESTIMATED $16.4 BILLION FOR A LIFETIME OF HIV TREATMENT FOR PATIENTS IN THE U.S. HIV INFECTS IMMUNE CELLS WHICH EXPRESS THE CD4 AND CXCR4/CCR5 CO-RECEPTORS INCLUDING HELPER T-CELLS. EVEN AFTER A PATIENT MOUNTS AN INITIAL, EFFECTIVE IMMUNE RESPONSE, THE VIRUS PERSISTS UNDETECTED BY THE IMMUNE SYSTEM IN QUIESCENTLY INFECTED CD4+ T-CELLS. FOR THIS REASON, THE PRIMARY TREATMENT FOR HIV, COMBINED ANTIRETROVIRAL THERAPY (CART), IS NOT ABLE TO ERADICATE HIV FROM PATIENTS. MANY HIV PATIENTS ARE ABLE TO LIVE WITH CONTINUOUS TREATMENT, BUT THIS IS BOTH COSTLY AND UNCERTAIN TO REMAIN EFFECTIVE GIVEN THE RECENT IDENTIFICATION OF A MORE VIRULENT STRAIN OF HIV-1 IN WHICH PATIENTS SUFFER AN ACCELERATED LOSS OF CD4+ T-CELLS. THERE IS A CRITICAL NEED FOR INNOVATIVE, EFFICACIOUS THERAPEUTICS TO DIRECT A MORE ROBUST IMMUNE ATTACK OF THE VIRUS. ONE PROMISING STRATEGY IS TO GENERATE MORE ACTIVE ANTI-HIV CD8+ CELLS. CHIMERIC ANTIGEN RECEPTOR-T CELL (CAR- T) IS A RELATIVELY NEW PROCESS IN WHICH PATIENT T-CELLS CAN BE PROGRAMMED TO ATTACK CELLS EXPRESSING A TARGET PROTEIN CHARACTERISTIC OF A SPECIFIC DISEASE. WHEN USED WITH AGENTS TO MAKE INFECTED CELLS VISIBLE, IT HAS BEEN HYPOTHESIZED THAT CAR-T WILL SUPPORT THE HOST IMMUNE SYSTEM TO FULLY ERADICATE HIV-INFECTED CELLS. THERE ARE CURRENTLY SEVERAL TECHNOLOGICAL LIMITATIONS TO THE PRODUCTION OF CAR-T CELLS, AND THE RATE LIMITING STEP IS TRANSFER OF GENETIC MATERIAL INTO THE PRIMARY T-CELLS TO PROGRAM THEM TO ELIMINATE CELLS EXPRESSING A TARGET PROTEIN. CURRENT METHODS OF CREATING CAR-T CELLS DEMONSTRATE AN INEFFICIENCY BARRING TRANSLATION FROM THE LABORATORY TO CLINICAL SETTINGS. LIPOFECTION INEFFICIENTLY TRANSFECTS PRIMARY T-CELLS, AND ELECTROPORATION AND BIOLISTICS BOTH DAMAGE CELLS. RETROVIRUSES ARE LIMITED TO 8-10 KB OF GENETIC MATERIAL WHICH LIMITS ADVANCED APPLICATIONS, ARE TOXIC TO CELLS IF USED AT TOO HIGH A DOSE, AND ARE COMPLEX TO CONSTRUCT. IN ADDITION, RETROVIRUSES INTEGRATE INTO THEIR TARGET CELL’S GENOME WHICH COULD INACTIVATE A TUMOR SUPPRESSOR GENE AND CREATE TUMORS. AGTC HAS DEVELOPED A NOVEL METHOD OF INTRODUCING BIOMOLECULES INTO MAMMALIAN CELLS USING AN ARRAY OF CLOSELY PACKED, ALIGNED CARBON NANOTUBES TO ACHIEVE HIGHLY EFFICIENT TRANSFER WITH LOW CYTOTOXICITY AND HIGH CAPACITY FOR GENETIC CARGO. THIS TECHNOLOGY HAS POTENTIAL TO OVERCOME SIZE LIMITS OF CURRENT GENE-TRANSFER TECHNOLOGIES IN ADDITION TO BEING SIMPLER, FASTER, AND MORE FLEXIBLE. IN THIS PROPOSAL, AGTC WILL (1) OPTIMIZE GENE TRANSFER INTO PRIMARY HUMAN T-CELLS USING CARBON NANOTUBE TECHNOLOGY (CNT); AND (2) CREATE HUMAN ANTI-HIV CD8+ CAR-T CELLS AGAINST HIV ENVELOPE GLYCOPROTEIN, GP120. SUCCESSFUL COMPLETION OF THE PROPOSED WORK WILL ENHANCE KNOWLEDGE ABOUT CNT CAPABILITIES, AND ALLOW AGTC TO PROCEED TO FURTHER TESTING OF THE CAR-T CELL PRODUCT WITH AN EYE TO THERAPEUTIC USE IN HIV POSITIVE PATIENTS.

273754.00

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