BACKGROUNDTransthyretin amyloidosis, also called ATTR amyloidosis, is a life-threatening disease characterized by progressive accumulation of misfolded transthyretin (TTR) protein in tissues, predominantly the nerves and heart. NTLA-2001 is an in vivo gene-editing therapeutic agent that is designed to treat ATTR amyloidosis by reducing the concentration of TTR in serum. It is based on the clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9) system and comprises a lipid nanoparticle encapsulating messenger RNA for Cas9 protein and a single guide RNA targeting TTR. METHODSAfter conducting preclinical in vitro and in vivo studies, we evaluated the safety and pharmacodynamic effects of single escalating doses of NTLA-2001 in six patients with hereditary ATTR amyloidosis with polyneuropathy, three in each of the two initial dose groups (0.1 mg per kilogram and 0.3 mg per kilogram), within an ongoing phase 1 clinical study. RESULTSPreclinical studies showed durable knockout of TTR after a single dose. Serial assessments of safety during the first 28 days after infusion in patients revealed few adverse events, and those that did occur were mild in grade. Dose-dependent pharmacodynamic effects were observed. At day 28, the mean reduction from baseline in serum TTR protein concentration was 52% (range, 47 to 56) in the group that received a dose of 0.1 mg per kilogram and was 87% (range, 80 to 96) in the group that received a dose of 0.3 mg per kilogram. CONCLUSIONSIn a small group of patients with hereditary ATTR amyloidosis with polyneuropathy, administration of NTLA-2001 was associated with only mild adverse events and led to decreases in serum TTR protein concentrations through targeted knockout of TTR. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.

This conditioning regimen prevented hyperacute rejection but was ineffective in preventing the return of Ab, which was associated with the development of acute humoral rejection with features of coagulopathy. No baboon developed anti-pig Ab other than alphaGal Ab. Further modifications of the protocol directed toward suppression of production of Ab are required to successfully induce tolerance to pig organs in baboons.

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Xenotransplantation could overcome the severe shortage of allogeneic organs, a major factor limiting organ transplantation. Unfortunately, transplantation of organs from pigs, the most suitable potential donor species, results in hyperacute rejection in primate recipients, due to the presence of anti–Galα1-3Gal (Gal) natural antibodies (NAbs) in their sera. We evaluated the ability to tolerize anti-Gal NAb–producing B cells in α1,3-galactosyltransferase knockout (GalT KO) mice using bone marrow transplantation (BMT) from GalT+/+ wild-type (WT) mice. Lasting mixed chimerism was achieved in KO mice by cotransplantation of GalT KO and WT marrow after lethal irradiation. The levels of anti-Gal NAb in sera of mixed chimeras were reduced markedly 2 wk after BMT, and became undetectable at later time points. Immunization with Gal+/+ xenogeneic cells failed to stimulate anti-Gal antibody production in mixed chimeras, whereas the production of non–Gal-specific antixenoantigen antibodies was stimulated. An absence of anti-Gal–producing B cells was demonstrated by enzyme-linked immunospot assays in mixed KO+WT→ KO chimeras. Thus, mixed chimerism efficiently induces anti-Gal–specific B cell tolerance in addition to T cell tolerance, providing a single approach to overcoming both the humoral and the cellular immune barriers to discordant xenotransplantation.

Human natural Abs against Galα1-3Galβ1-4GlcNAc (Gal) epitopes are a major barrier to xenotransplantation. Studies in this report, which use combined multiparameter flow cytometric sorting and enzyme-linked immunospot assay, demonstrate that anti-Gal IgM-producing cells are found exclusively in a small B cell subpopulation (i.e., CD21−/low IgMhigh B220low CD5− Mac-1− 493− cells) in the spleens of α1,3-galactosyltransferase-deficient mice. All IgM-producing cells were detected in a similar splenic subpopulation of α1,3-galactosyltransferase-deficient and wild-type mice. A higher frequency of B cells with anti-Gal surface IgM receptors was observed in the peritoneal cavity than in the spleen, but these did not actively secrete Abs, and showed phenotypic properties of B-1b cells (CD21−/low IgMhigh CD5− CD43+ Mac-1+). However, these became Mac-1− and developed anti-Gal Ab-producing activity after in vitro culture with LPS. The splenic B cells with anti-Gal receptors consisted of both Mac-1+ B-1b cells and Mac-1− B-1b-like cells. The latter comprised most anti-Gal IgM-producing cells. Our studies indicate that anti-Gal natural IgM Abs are produced by a B1b-like, Mac-1− splenic B cell population and not by plasma cells or B-1a cells. They are consistent with a model whereby B-1b cells lose Mac-1 expression upon Ag exposure and that these, rather than plasma cells, become the major IgM Ab-producing cell population.

Both methods reduced the level of anti-pig IgM and IgG xenoreactive antibodies to nearly background, but column perfusion caused less hypotension and reduction in platelets than liver perfusion. Four pig kidneys transplanted into monkeys after column perfusion did not undergo hyperacute rejection, remaining functional for 2-10 days, with a mean functional period of 7 days, demonstrating that a pig kidney can support renal function in a primate.

In the present study, we assessed the feasibility, toxicity, immunologic response, and clinical efficacy of vaccination with allogeneic dendritic cell (DC)/tumor fusions in patients with metastatic renal cell carcinoma (RCC). Patients with stage IV RCC with accessible tumor lesions or independent therapeutic indications for nephrectomy were eligible for enrollment. Tumors were processed into single cell suspensions and cryopreserved. DCs were generated from adherent peripheral blood mononuclear cells isolated from normal volunteers and cultured with granulocyte macrophage colony-stimulating factor, interleukin-4, and tumor necrosis factor-alpha. DCs were fused to patient derived RCC with serial electrical pulses. Patients received up to 3 vaccinations at a fixed dose of 4x10(7) to 1x10(8) cells administered at 6-week intervals. Twenty-four patients underwent vaccination. Twenty-one and 20 patients were evaluable for immunologic and clinical response, respectively. DCs demonstrated a characteristic phenotype with prominent expression of HLA class II and costimulatory molecules. A mean fusion efficiency of 20% was observed, determined by the percent of cells coexpressing DC and tumor antigens. No evidence of significant treatment related toxicity or auto-immunity was observed. Vaccination resulted in antitumor immune responses in 10/21 evaluable patients as manifested by an increase in CD4 and/or CD8 T-cell expression of interferon-gamma after ex vivo exposure to tumor lysate. Two patients demonstrated a partial clinical response by Response Evaluation Criteria in Solid Tumors criteria and 8 patients had stabilization of their disease. Vaccination of patients with RCC with allogeneic DC/tumor fusions was feasible, well tolerated, and resulted in immunologic and clinical responses in a subset of patients.

interfering ribonucleic acid; siRNA1-siRNA22, 22 distinct siRNAs used in the in vitro and in vivo studies with data presented in this work; STC, standard template chemistry; t 1/2 , half-life; T max , time at which maximal concentration was observed.