Precision targeting of bladder cancer using CRISPR

Document text

Principal Investigator: Mats  Ljungman
Organization: UNIVERSITY OF MICHIGAN AT ANN ARBOR
Fiscal Year: 2024
Award: $566,310
Funding agency: National Cancer Institute

ABSTRACT
Chromosome rearrangements (structural variants) are common in cancer and they drive tumor
development by altering the expression or function of oncogenes and tumor suppressor genes
by copy number alterations, formation of oncogenic fusions or by alterations of DNA elements
responsible for regulating cancer genes. We have developed an innovative CRISPR-based
approach to specifically target structural variant junctions (SVJs) in cancer cells without harming
normal cells. This approach, called KLIPP, is based on the use of a “split” enzyme approach
consisting of inactivated dCas9 fused to the endonuclease Fok1 (Fok1-dCas9). To activate the
Fok1 endonuclease, two Fok1-dCas9 complexes are brought together at the SVJ using SVJ-
targeting guide RNAs leading to the induction of toxic DNA double strand breaks. We have
obtained strong proof-of-concept for this approach in bladder cancer and other cancer cell
systems both in cell cultures and in vivo. In this proposal we will perform pre-clinical efficacy
testing of the Precision KLIPP Therapy approach in an orthotopic mouse model of bladder
cancer. The premise for targeting bladder cancer with this approach is first, locally invasive
bladder cancer is a common and deadly disease with few impactful therapeutic options.
Second, bladder cancers typically show genomic instability with many hundreds of SVJs that
could be targeted with our approach. Third, the bladder is ideal for local inter-vesicle delivery of
the CRISPR reagents because of the ability to achieve high local concentrations and avoiding
systemic effects. We will in this application pursue three specific aims: Aim #1: CRISPR screen
to map potential Fok1-dCas9 binding toxicity genome-wide. Aim #2: Delivery of CRISPR
reagents to bladder cells in culture. Aim #3: Assessment of efficacy of Precision KLIPP Therapy
in a pre-clinical HGSC mouse model. With this R01 funding, we will be able to advance this
project to the point of planning for human clinical trials.

Terms: <Animal Model><Animal Models and Related Studies><Anti-Oncogenes><Antioncogenes><Apoptosis><Apoptosis Pathway><Binding><Bio-Informatics><Bioinformatics><Biology><Bladder><Bladder Cancer><Bladder Neoplasm><Bladder Tumors><Bladder Urinary System><CRISPR><CRISPR approach><CRISPR based approach><CRISPR editing screen><CRISPR method><CRISPR methodology><CRISPR screen><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based screen><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas system><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 screen><CRISPR/Cas9 technology><Cancer Biology><Cancer Genes><Cancer Model><Cancer Suppressor Genes><Cancer Treatment><Cancer cell line><Cancer-Promoting Gene><CancerModel><Cancers><Cas nuclease technology><Cell Body><Cell Culture Techniques><Cell Death><Cell Line><CellLine><Cells><Chromosomes><Circulation><Clinical><Clinical Trials><Clustered Regularly Interspaced Short Palindromic Repeats><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Complex><DNA Alteration><DNA Double Strand Break><DNA Sequence Alteration><DNA mutation><Development><Dimerization><Disease><Disorder><Disseminated Malignant Neoplasm><Elements><Emerogenes><Encapsulated><Enzyme Gene><Enzymes><Essential Genes><Funding><Gamma-H2AX><Gene Copy Number><Gene Dosage><Generalized Growth><Genetic mutation><Genome><Genome Instability><Genomic Instability><Genomics><Goals><Growth><Guide RNA><Human><Image><Investigators><Libraries><Malignant Bladder Neoplasm><Malignant Cell><Malignant Neoplasm Therapy><Malignant Neoplasm Treatment><Malignant Neoplasms><Malignant Tumor><Malignant Tumor of the Bladder><Malignant neoplasm of urinary bladder><Maps><Medicine><Messenger RNA><Metastatic Cancer><Metastatic Malignant Neoplasm><Mice><Mice Mammals><Modeling><Modern Man><Molecular Interaction><Monitor><Murine><Mus><Normal Cell><Onco-Suppressor Genes><Oncogene Activation><Oncogenes><Oncogenes-Tumor Suppressors><Oncogenic><Patients><Process><Programmed Cell Death><Protein Dimerization><Reagent><Recessive Oncogenes><Research Personnel><Researchers><Sequence Alteration><Strains Cell Lines><System><Technology><Testing><Therapeutic><Time><Tissue Growth><Toxic effect><Toxicities><Transforming Genes><Tumor Biology><Tumor Cell><Tumor Suppressing Genes><Tumor Suppressor Genes><Urinary Bladder Cancer><Urinary Bladder Malignant Tumor><Urinary Bladder Neoplasm><Urinary Bladder Tumor><Variant><Variation><Vesicle><Work><anti-cancer therapy><cancer cell><cancer therapy><cancer-directed therapy><carcinogenicity><cell culture><cell cultures><clustered regularly interspaced short palindromic repeats screen><cultured cell line><design><designing><determine efficacy><developmental><efficacy analysis><efficacy assessment><efficacy determination><efficacy evaluation><efficacy examination><efficacy testing><endonuclease><evaluate efficacy><examine efficacy><gRNA><genome scale><genome-wide><genomewide><genomic alteration><imaging><in vivo><innovate><innovation><innovative><lipid based nanoparticle><lipid nanoparticle><mRNA><malignancy><manufacture><model of animal><mouse model><murine model><nano particle delivery><nanoparticle delivered><nanoparticle delivery><necrocytosis><neoplasm/cancer><neoplastic cell><oncosuppressor gene><ontogeny><particle><pre-clinical><pre-clinical efficacy><preclinical><preclinical efficacy><resistance mechanism><resistant mechanism><technology platform><technology system><tumor><urinary bladder><γH2AX>