RESUMO
Monogenic blood diseases are among the most common genetic disorders worldwide. These diseases result in significant pediatric and adult morbidity, and some can result in death prior to birth. Novel ex vivo hematopoietic stem cell (HSC) gene editing therapies hold tremendous promise to alter the therapeutic landscape but are not without potential limitations. In vivo gene editing therapies offer a potentially safer and more accessible treatment for these diseases but are hindered by a lack of delivery vectors targeting HSCs, which reside in the difficult-to-access bone marrow niche. Here, we propose that this biological barrier can be overcome by taking advantage of HSC residence in the easily accessible liver during fetal development. To facilitate the delivery of gene editing cargo to fetal HSCs, we developed an ionizable lipid nanoparticle (LNP) platform targeting the CD45 receptor on the surface of HSCs. After validating that targeted LNPs improved messenger ribonucleic acid (mRNA) delivery to hematopoietic lineage cells via a CD45-specific mechanism in vitro, we demonstrated that this platform mediated safe, potent, and long-term gene modulation of HSCs in vivo in multiple mouse models. We further optimized this LNP platform in vitro to encapsulate and deliver CRISPR-based nucleic acid cargos. Finally, we showed that optimized and targeted LNPs enhanced gene editing at a proof-of-concept locus in fetal HSCs after a single in utero intravenous injection. By targeting HSCs in vivo during fetal development, our Systematically optimized Targeted Editing Machinery (STEM) LNPs may provide a translatable strategy to treat monogenic blood diseases before birth.
Assuntos
Edição de Genes , Células-Tronco Hematopoéticas , Nanopartículas , Animais , Células-Tronco Hematopoéticas/metabolismo , Edição de Genes/métodos , Nanopartículas/química , Camundongos , Feminino , Gravidez , Lipídeos/química , Antígenos Comuns de Leucócito/metabolismo , Antígenos Comuns de Leucócito/genética , Humanos , Terapia Genética/métodos , Sistemas CRISPR-Cas , LipossomosRESUMO
BACKGROUND: Our team has previously reported physiologic support by the EXTra-uterine Environment for Neonatal Development (EXTEND) of 105 to 117 days gestational age (GA) lambs for up to 28 days with normal organ maturation. However, the fetal lamb brain matures more rapidly, requiring the study of 90-105 day GA fetal lambs to assess more neurodevelopmentally equivalent lambs to the 23-25 week GA extreme premature infant. METHODS: Extremely preterm lambs (90-95 days of GA) were delivered by C-section and supported by EXTEND. Estimated circuit flows were maintained at around 325 ml/kg/min. After support on EXTEND, MRI and histopathologic analysis were performed and compared to 105-112 days GA control lambs. RESULTS: The extremely preterm group includes 10 animals with a mean GA of 91.6 days, a mean weight at cannulation of 0.98 kg and a mean length of stay on EXTEND of 13.5 days (10-21 days). Hemodynamics and oxygenation showed stable parameters. Animals showed growth and physiologic cardiac function. MRI volumetric and diffusion analysis was comparable to controls. Histologic brain analysis revealed no difference between study groups. CONCLUSION: EXTEND appears to support brain and cardiac development in an earlier gestation, less mature, lamb model. IMPACT: Prolonged (up to 21 days) physiological support of extremely preterm lambs of closer neurodevelopmental equivalence to the 24-28 gestational week human was achieved using the EXTEND system. EXTEND treatment supported brain growth and development in extremely preterm fetal lambs and was not associated with intraventricular hemorrhage or white matter injury. Daily echocardiography demonstrated physiologic heart function, absence of cardiac afterload, and normal developmental increase in cardiac chamber dimensions. This study demonstrates hemodynamic and metabolic support by the EXTEND system in the extremely preterm ovine model.
Assuntos
Cuidado Pré-Natal , Gravidez , Feminino , Humanos , Suínos , Animais , Animais Recém-NascidosRESUMO
MicroRNAs (miRNAs) and siRNAs have enormous potential as cancer therapeutics, but their effective delivery to most solid tumors has been difficult. Here, we show that a new lung-targeting nanoparticle is capable of delivering miRNA mimics and siRNAs to lung adenocarcinoma cells in vitro and to tumors in a genetically engineered mouse model of lung cancer based on activation of oncogenic Kirsten rat sarcoma viral oncogene homolog (Kras) and loss of p53 function. Therapeutic delivery of miR-34a, a p53-regulated tumor suppressor miRNA, restored miR-34a levels in lung tumors, specifically down-regulated miR-34a target genes, and slowed tumor growth. The delivery of siRNAs targeting Kras reduced Kras gene expression and MAPK signaling, increased apoptosis, and inhibited tumor growth. The combination of miR-34a and siRNA targeting Kras improved therapeutic responses over those observed with either small RNA alone, leading to tumor regression. Furthermore, nanoparticle-mediated small RNA delivery plus conventional, cisplatin-based chemotherapy prolonged survival in this model compared with chemotherapy alone. These findings demonstrate that RNA combination therapy is possible in an autochthonous model of lung cancer and provide preclinical support for the use of small RNA therapies in patients who have cancer.
Assuntos
Neoplasias Pulmonares/terapia , MicroRNAs/uso terapêutico , RNA Interferente Pequeno/uso terapêutico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/terapia , Animais , Antineoplásicos/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/terapia , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Terapia Combinada , Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Camundongos Transgênicos , MicroRNAs/administração & dosagem , MicroRNAs/genética , Mutação , Nanopartículas/administração & dosagem , Nanopartículas/uso terapêutico , Nanotecnologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteínas ras/genéticaRESUMO
In utero hematopoietic cell transplantation (IUHCT) is an experimental non-myeloablative therapy with potential application to hematologic disorders including Sickle cell disease. Its clinical utility has been limited due to the early acquisition of T cell immunity beginning at approximately 14 weeks gestation, posing significant technical challenges and excluding from treatment fetuses evaluated after the first trimester. Using murine neonatal transplantation at 20 days post-coitum (DPC) as a model for late-gestation transplantation (LGT) in humans, we investigated whether immune modulation with anti-CD3 monoclonal antibody (mAb) could achieve donor-specific tolerance and sustained allogeneic engraftment comparable to the early-gestation fetal recipient at 14 DPC. In allogeneic wild-type strain combinations, administration of anti-CD3 mAb with transplantation resulted in transient T cell depletion followed by central tolerance induction confirmed by donor-specific clonal deletion and skin graft tolerance. Normal immune responses to third-party major histocompatibility complex and viral pathogens were preserved, and graft-versus-host disease did not occur. We further demonstrate successful application of this approach to the Townes mouse model of Sickle cell disease. These findings confirm the developing fetal T cell response as a barrier to LGT and support transient T cell depletion as a safe and effective immunomodulatory strategy by which to overcome it.
RESUMO
In utero gene editing (IUGE) is a potential treatment for inherited diseases that cause pathology before or soon after birth. Preexisting immunity to adeno-associated virus (AAV) vectors and Cas9 endonuclease may limit postnatal gene editing. The tolerogenic fetal immune system minimizes a fetal immune barrier to IUGE. However, the ability of maternal immunity to limit fetal gene editing remains a question. We investigated whether preexisting maternal immunity to AAV or Cas9 impairs IUGE. Using a combination of fluorescent reporter mice and a murine model of a metabolic liver disease, we demonstrated that maternal anti-AAV IgG antibodies were efficiently transferred from dam to fetus and impaired IUGE in a maternal titer-dependent fashion. By contrast, maternal cellular immunity was inefficiently transferred to the fetus, and neither maternal cellular nor humoral immunity to Cas9 impaired IUGE. Using human umbilical cord and maternal blood samples collected from mid- to late-gestation pregnancies, we demonstrated that maternal-fetal transmission of anti-AAV IgG was inefficient in midgestation compared with term, suggesting that the maternal immune barrier to clinical IUGE would be less relevant at midgestation. These findings support immunologic advantages for IUGE and inform maternal preprocedural testing protocols and exclusion criteria for future clinical trials.
Assuntos
Dependovirus , Edição de Genes , Animais , Feminino , Dependovirus/genética , Dependovirus/imunologia , Camundongos , Gravidez , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/genética , Imunoglobulina G/sangue , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/imunologia , Vetores Genéticos/imunologia , Troca Materno-Fetal/imunologia , Troca Materno-Fetal/genética , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Sistemas CRISPR-Cas , Feto/imunologia , Imunidade Materno-Adquirida/imunologiaRESUMO
In utero hematopoietic cell transplantation (IUHCT) is an experimental treatment for congenital hemoglobinopathies, including Sickle cell disease and thalassemias. One of the principal advantages of IUHCT is the predisposition of the developing fetus toward immunologic tolerance. This allows for engraftment across immune barriers without immunosuppression and, potentially, decreased susceptibility to graft-versus-host disease (GVHD). We demonstrate fetal resistance to GVHD following T cell-replete allogeneic hematopoietic cell transplantation compared with the neonate. We show that this resistance is associated with elevated fetal serum interleukin-10 conducive to the induction of regulatory T cells (Tregs). Finally, we demonstrate that the adoptive transfer of Tregs from IUHCT recipients to neonates uniformly prevents GVHD, recapitulating the predisposition to tolerance observed after fetal allotransplantation. These findings demonstrate fetal resistance to GVHD following hematopoietic cell transplantation and elucidate Tregs as important contributors.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Humanos , Recém-Nascido , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Tolerância Imunológica , Feto , Linfócitos T ReguladoresRESUMO
Delivery of mRNA-based therapeutics to the perinatal brain holds great potential in treating congenital brain diseases. However, nonviral delivery platforms that facilitate nucleic acid delivery in this environment have yet to be rigorously studied. Here, we screen a diverse library of ionizable lipid nanoparticles (LNPs) via intracerebroventricular (ICV) injection in both fetal and neonatal mice and identify an LNP formulation with greater functional mRNA delivery in the perinatal brain than an FDA-approved industry standard LNP. Following in vitro optimization of the top-performing LNP (C3 LNP) for codelivery of an adenine base editing platform, we improve the biochemical phenotype of a lysosomal storage disease in the neonatal mouse brain, exhibit proof-of-principle mRNA brain transfection in vivo in a fetal nonhuman primate model, and demonstrate the translational potential of C3 LNPs ex vivo in human patient-derived brain tissues. These LNPs may provide a clinically translatable platform for in utero and postnatal mRNA therapies including gene editing in the brain.
Assuntos
Encefalopatias , Nanopartículas , Camundongos , Humanos , Animais , Edição de Genes , Lipídeos , Lipossomos , RNA Mensageiro/genética , RNA Interferente Pequeno/genéticaRESUMO
Congenital disorders resulting in pathological protein deficiencies are most often treated postnatally with protein or enzyme replacement therapies. However, treatment of these disorders in utero before irreversible disease onset could significantly minimize disease burden, morbidity, and mortality. One possible strategy for the prenatal treatment of congenital disorders is in utero delivery of messenger RNA (mRNA). mRNA is a nucleic acid therapeutic that has previously been investigated as a platform for protein replacement therapies and gene editing technologies. While viral vectors have been explored to induce intracellular expression of mRNA, they are limited in their clinical application due to risks associated with immunogenicity and genomic integration. As an alternative to viral vectors, safe and efficient in utero mRNA delivery can be achieved using ionizable lipid nanoparticles (LNPs). While LNPs have demonstrated potent in vivo mRNA delivery to the liver following intravenous administration, intra-amniotic delivery has the potential to deliver mRNA to cells and tissues beyond those in the liver, such as in the skin, lung, and digestive tract. However, LNP stability in fetal amniotic fluid and how this stability affects mRNA delivery has not been previously investigated. Here, we engineered a library of LNPs using orthogonal design of experiments (DOE) to evaluate how LNP structure affects their stability in amniotic fluid ex utero and whether a lead candidate identified from these stability measurements enables intra-amniotic mRNA delivery in utero. We used a combination of techniques including dynamic light scattering (DLS), transmission electron microscopy (TEM), and chromatography followed by protein content quantification to screen LNP stability in amniotic fluids. These results identified multiple lead LNP formulations that are highly stable in amniotic fluids ranging from small animals to humans, including mouse, sheep, pig, and human amniotic fluid samples. We then demonstrate that stable LNPs from the ex utero screen in mouse amniotic fluid enabled potent mRNA delivery in primary fetal lung fibroblasts and in utero following intra-amniotic injection in a murine model. This exploration of ex utero stability in amniotic fluids demonstrates a means by which to identify novel LNP formulations for prenatal treatment of congenital disorders via in utero mRNA delivery.
Assuntos
Líquido Amniótico , Nanopartículas , Animais , Lipossomos/química , Camundongos , Nanopartículas/química , RNA Mensageiro , Ovinos , SuínosRESUMO
BACKGROUND: The utilization of cancer-directed treatment for patients with all stages of pancreatic cancer in the USA is unknown. This study sought to examine national practice patterns and identify patient, hospital, regional, and other factors associated with disparities in the use of guideline-concordant cancer-directed therapy. METHODS: Patients diagnosed with PDAC between 2004 and 2015 were queried from the National Cancer Data Base. Standard of care cancer-directed treatment was defined as surgical resection plus chemotherapy or chemoradiation for patients with stage 1 and 2 disease, chemotherapy for patients with metastatic disease (stage 4), and chemotherapy with or without surgery or chemoradiation for patients with locally advanced stage 3 disease. RESULTS: A total of 336,629 patients with stage 1 (n = 38,443, 11.4%), stage 2 (n = 93,923, 27.9%), stage 3 (n = 37,492, 11.1%), or stage 4 metastatic (n = 166,771, 49.5%) disease were identified. Adherence with stage-specific standard of care treatment occurred in only 45.3% (n = 152,560) of patients among the entire cohort and varied by stage of disease (stage 1: 14.6% vs. stage 2: 39.9% vs. stage 3: 67.6%, vs. stage 4: 50.9%). Older age (OR 0.95, 95%CI 0.94-0.95; p < 0.001), female sex (OR 0.94, 95%CI 0.943-0.97; p < 0.001), African Americans (OR 0.89, 95%CI 0.87-0.91; P < 0.001), and increasing comorbidity burden (Charlson-Deyo score ≥3: OR 0.52, 95%CI 0.50-0.55; P < 0.001) were associated with a lower likelihood of receiving stage-specific standard of care treatment. Conversely, treatment at a high-volume center (quartile 4: OR: 1.13, 95%CI 1.10-1.16; P < 0.001) and higher education level (OR 1.32, 95%CI 1.28-1.36; p < 0.001) was associated with higher likelihood of receiving stage-specific standard of care treatment. Patients who received standard of care treatment had a 47% lower risk of death compared with patients who did not receive standard of care treatment (HR 0.53, 95%CI 0.52-0.53; P < 0.001). CONCLUSION: Pancreatic adenocarcinoma is a complex disease requiring a multi-disciplinary approach for optimal outcomes. Receipt of stage-specific standard of care treatment for PDAC is associated with improved long-term oncological outcomes, but is only achieved in less than half of patients. Further studies are needed to evaluate interventions to address these treatment disparities for patients with PDAC.
Assuntos
Adenocarcinoma , Neoplasias Pancreáticas , Adenocarcinoma/tratamento farmacológico , Negro ou Afro-Americano , Idoso , Quimiorradioterapia , Bases de Dados Factuais , Feminino , Disparidades em Assistência à Saúde , Humanos , Neoplasias Pancreáticas/tratamento farmacológicoRESUMO
In utero base editing has the potential to correct disease-causing mutations before the onset of pathology. Mucopolysaccharidosis type I (MPS-IH, Hurler syndrome) is a lysosomal storage disease (LSD) affecting multiple organs, often leading to early postnatal cardiopulmonary demise. We assessed in utero adeno-associated virus serotype 9 (AAV9) delivery of an adenine base editor (ABE) targeting the Idua GâA (W392X) mutation in the MPS-IH mouse, corresponding to the common IDUA GâA (W402X) mutation in MPS-IH patients. Here we show efficient long-term W392X correction in hepatocytes and cardiomyocytes and low-level editing in the brain. In utero editing was associated with improved survival and amelioration of metabolic, musculoskeletal, and cardiac disease. This proof-of-concept study demonstrates the possibility of efficiently performing therapeutic base editing in multiple organs before birth via a clinically relevant delivery mechanism, highlighting the potential of this approach for MPS-IH and other genetic diseases.
Assuntos
Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/patologia , Animais , Modelos Animais de Doenças , Hepatócitos/metabolismo , Humanos , Mutação/genética , Miócitos Cardíacos/metabolismoRESUMO
BACKGROUND: In a changing health care environment where patient outcomes will be more closely scrutinized, the ability to predict surgical complications is becoming increasingly important. The American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) online risk calculator is a popular tool to predict surgical risk. This paper aims to assess the applicability of the ACS NSQIP calculator to patients undergoing surgery for pancreatic neuroendocrine tumors (PNETs). METHODS: Using the US Neuroendocrine Tumor Study Group (USNET-SG), 890 patients who underwent pancreatic procedures between 1/1/2000-12/31/2016 were evaluated. Predicted and actual outcomes were compared using C-statistics and Brier scores. RESULTS: The most commonly performed procedure was distal pancreatectomy, followed by standard and pylorus-preserving pancreaticoduodenectomy. For the entire group of patients studied, C-statistics were highest for discharge destination (0.79) and cardiac complications (0.71), and less than 0.7 for all other complications. The Brier scores for surgical site infection (0.1441) and discharge to nursing/rehabilitation facility (0.0279) were below the Brier score cut-off, while the rest were equal to or above and therefore not useful for interpretation. CONCLUSION: This work indicates that the ACS NSQIP risk calculator is a valuable tool that should be used with caution and in coordination with clinical assessment for PNET clinical decision-making.
Assuntos
Tumores Neuroendócrinos/cirurgia , Neoplasias Pancreáticas/cirurgia , Pancreaticoduodenectomia/métodos , Complicações Pós-Operatórias/epidemiologia , Melhoria de Qualidade , Medição de Risco/métodos , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
BACKGROUND/PURPOSE: A tissue-engineered esophagus offers an alternative for the treatment of pediatric patients suffering from severe esophageal malformations, caustic injury, and cancer. Additionally, adult patients suffering from carcinoma or trauma would benefit. METHODS: Donor rat esophageal tissue was physically and enzymatically digested to isolate epithelial and smooth muscle cells, which were cultured in epithelial cell medium or smooth muscle cell medium and characterized by immunofluorescence. Isolated cells were also seeded onto electrospun synthetic PLGA and PCL/PLGA scaffolds in a physiologic hollow organ bioreactor. After 2 weeks of in vitro culture, tissue-engineered constructs were orthotopically transplanted. RESULTS: Isolated cells were shown to give rise to epithelial, smooth muscle, and glial cell types. After 14 days in culture, scaffolds supported epithelial, smooth muscle and glial cell phenotypes. Transplanted constructs integrated into the host's native tissue and recipients of the engineered tissue demonstrated normal feeding habits. Characterization after 14 days of implantation revealed that all three cellular phenotypes were present in varying degrees in seeded and unseeded scaffolds. CONCLUSIONS: We demonstrate that isolated cells from native esophagus can be cultured and seeded onto electrospun scaffolds to create esophageal constructs. These constructs have potential translatable application for tissue engineering of human esophageal tissue.
Assuntos
Esôfago/citologia , Esôfago/transplante , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Biomimética/métodos , Reatores Biológicos , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/transplante , Feminino , Ácido Láctico/química , Masculino , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/transplante , Poliésteres/química , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ratos , Ratos Sprague-DawleyRESUMO
Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis.