Jack Castelli

All Publications

• In vivo production of an anti-HIV antibody in mice by non-viral gene knock-in in primate hematopoietic stem and progenitor cells. Molecular therapy : the journal of the American Society of Gene Therapy Castelli, J. M., Poljakov, K., Jwa, Y., Cunningham, R., Cassidy, M. E., Gray, M. D., Sanchez Gaytan, J. N., Enstrom, M. R., Gastelum, G., Wang, Z., Linton, J. D., Rongvaux, A., Taylor, J. J., Adair, J. E. 2026

  Abstract

  Gene editing strategies that do not rely on viral vectors are being explored for their potential to support durable biologics production. While clinical trials have shown that adeno-associated virus encoding broadly neutralizing antibodies can protect against HIV, these interventions often yield limited, short-lived responses. The development of non-viral gene editing approaches in hematopoietic stem and progenitor cells holds promise for long-term antibody production. In this study, we evaluated CRISPR/Cas9 and CRISPR/Cas12a for gene knock-in at the immunoglobulin heavy chain locus in non-human primate (NHP) hematopoietic stem and progenitor cells (HSPCs). Delivering the nuclease as a protein alongside a custom DNA template, we optimized editing with Cas12a and demonstrated higher knock-in efficiency and fewer non-specific edits than Cas9. Transplantation of edited NHP HSPCs into MISTRG mice led to engraftment, B cell differentiation, and transgene expression of a reporter transgene or anti-HIV antibody after gp120 antigen immunization with detectable titers in circulation. These findings demonstrate the feasibility of using non-viral knock-in in HSPCs as a potential strategy for sustained biologics production in the treatment of chronic diseases. Future work will assess the efficacy of this approach in a NHP model of HIV infection.

  View details for DOI 10.1016/j.ymthe.2026.01.038

  View details for PubMedID 41635086

• CRISPR-AuNP: physicochemical optimization of a gold nanoparticle platform for cost-effective and modular non-viral gene editing in HSPCs. Gene therapy Gottimukkala, K. S., Lane, D. D., Cunningham, R., Malik, H. S., Jwa, Y., Cassidy, M. E., Castelli, J. M., Enstrom, M. R., Poljakov, K., Gastelum, G., Ho, S. H., Tassa, C., Adair, J. E. 2026

  Abstract

  Efficient delivery of CRISPR ribonucleoproteins into primary hematopoietic stem and progenitor cells (HSPCs) is essential for durable gene editing therapies but remains challenging. Here, we advance a modular, benchtop-assembled gold-polymer hybrid nanoparticle (CRISPR-AuNP) platform that enables non-viral delivery of multiple CRISPR systems into HSPCs. Guided by a mechanistic understanding of Cas9's interaction with gold surfaces, we engineered the formulation by conjugating pre-formed RNP-polymer complexes, assembled using thiolated polyethyleneimine-polyethylene glycol, to gold nanoparticles. This system achieved efficient editing in primary CD34+ HSPCs for Cas9, Cas12a, and Cas12a-M29-1 without compromising cell viability. Notably, the nanoformulation can be assembled in under 2 h in a PCR tube for less than $70/million HSPCs treated. This work establishes a scalable, cost-effective, and accessible gene editing system with the potential to democratize CRISPR applications in HSPC research and therapy.

  View details for DOI 10.1038/s41434-025-00591-0

  View details for PubMedID 41535609

  View details for PubMedCentralID 6286148

• 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