Jack Castelli

All Publications

• The functional landscape of coding variation in the familial hypercholesterolemia gene LDLR. Science (New York, N.Y.) Tabet, D. R., Coté, A. G., Lancaster, M. C., Weile, J., Rayhan, A., Fotiadou, I., Kishore, N., Li, R., Kuang, D., Knapp, J. J., Carrero, C. S., Taverniti, O., Axakova, A., Castelli, J. M., Islam, M. M., Sowlati-Hashjin, S., Gandhi, A., Maaieh, R., Garton, M., Matreyek, K., Fowler, D. M., Bourbon, M., Pfisterer, S. G., Glazer, A. M., Kroncke, B. M., Parikh, V. N., Ashley, E. A., Knowles, J. W., Claussnitzer, M., Cirulli, E. T., Hegele, R. A., Roden, D. M., MacRae, C. A., Roth, F. P. 2025: eady7186

  Abstract

  Variants in the familial hypercholesterolemia gene LDLR-the most important genetic driver of cardiovascular disease-can raise circulating low-density lipoprotein (LDL) cholesterol concentrations and increase the risk of premature atherosclerosis. Definitive classifications are lacking for nearly half of clinically encountered LDLR missense variants, limiting interventions that reduce disease burden. Here, we tested the impact of ~17,000 (nearly all possible) LDLR missense coding variants on both LDLR cell-surface abundance and LDL uptake, yielding sequence-function maps that recapitulate known biochemistry, offer functional insights, and provide evidence for interpreting clinical variants. Functional scores correlated with hyperlipidemia phenotypes in prospective human cohorts and augmented polygenic scores to improve risk inference, highlighting the potential of this resource to accelerate familial hypercholesterolemia diagnosis and improve patient outcomes.

  View details for DOI 10.1126/science.ady7186

  View details for PubMedID 41166440

• Cyprocide selectively kills nematodes via cytochrome P450 bioactivation NATURE COMMUNICATIONS Knox, J., Burns, A. R., Cooke, B., Cammalleri, S. R., Kitner, M., Ching, J., Castelli, J. M. P., Puumala, E., Snider, J., Koury, E., Collins, J. B., Geissah, S., Dowling, J. J., Andersen, E. C., Stagljar, I., Cowen, L. E., Lautens, M., Zasada, I., Roy, P. J. 2024; 15 (1): 5529

  Abstract

  Left unchecked, plant-parasitic nematodes have the potential to devastate crops globally. Highly effective but non-selective nematicides are justifiably being phased-out, leaving farmers with limited options for managing nematode infestation. Here, we report our discovery of a 1,3,4-oxadiazole thioether scaffold called Cyprocide that selectively kills nematodes including diverse species of plant-parasitic nematodes. Cyprocide is bioactivated into a lethal reactive electrophilic metabolite by specific nematode cytochrome P450 enzymes. Cyprocide fails to kill organisms beyond nematodes, suggesting that the targeted lethality of this pro-nematicide derives from P450 substrate selectivity. Our findings demonstrate that Cyprocide is a selective nematicidal scaffold with broad-spectrum activity that holds the potential to help safeguard our global food supply.

  View details for DOI 10.1038/s41467-024-49738-4

  View details for Web of Science ID 001261751100013

  View details for PubMedID 38956039

  View details for PubMedCentralID PMC11219838

• Cas9 RNP Physiochemical Analysis for Enhanced CRISPR-AuNP Assembly and Function. bioRxiv : the preprint server for biology Lane, D. D., Gottimukkala, K. S., Cunningham, R. A., Jwa, S., Cassidy, M. E., Castelli, J. M., Adair, J. E. 2024

  Abstract

  CRISPR therapy for hematological disease has proven effective for transplant dependent beta thalassemia and sickle cell anemia, with additional disease targets in sight. The success of these therapies relies on high rates of CRISPR-induced double strand DNA breaks in hematopoietic stem and progenitor cells (HSPC). To achieve these levels, CRISPR complexes are typically delivered by electroporation ex vivo which is toxic to HSPCs. HSPCs are then cultured in stimulating conditions that promote error-prone DNA repair, requiring conditioning with chemotherapy to facilitate engraftment after reinfusion. In vivo delivery by nanocarriers of CRISPR gene editing tools has the potential to mitigate this complexity and toxicity and make this revolutionary therapy globally available. To achieve in vivo delivery, the inherent restriction factors against oligonucleotide delivery into HSPCs, that make ex vivo manipulation including electroporation and stimulation essential, must be overcome. To this end, our group developed a CRISPR carrying gold nanoparticle (CRISPR-AuNP) capable of delivering either Cas9 or Cas12a CRISPRs as ribonucleoprotein complexes (RNP) without compromising HSPC fitness. However, the most commonly used CRISPR, Cas9, demonstrated inconsistent activity in this delivery system, with lower activity relative to Cas12a. Investigation of Cas9 RNP biophysics relative to Cas12a revealed duplex RNA instability during the initial loading onto Au cores, resulting in undetectable Cas9 loading to the particle surface. Here we demonstrate preformation of RNP before loading, coupled with optimization of the loading chemistry and conditions, resulted in 39.6 ± 7.0 Cas9 RNP/AuNP without compromising RNP activity in both in vitro assays and primary human HSPC. The same alterations improved Cas12a RNP/AuNP loading 10-fold over previously reported levels. To achieve particle stability, the reported polyethyleneimine outer coating was altered to include PEGylation and the resulting 2nd generation CRISPR-AuNP demonstrates favorable nanoformulation characteristics for in vivo administration, with a hydrophilic, more neutral nanoparticle surface. Direct treatment of HSPC in vitro showed 72.5 ± 7.37% uptake of 2nd generation CRISPR-AuNP in primary human HSPC, but with endosomal accumulation and low rates of gene editing consistent with low levels of endosomal escape.

  View details for DOI 10.1101/2024.04.02.586657

  View details for PubMedID 38617334