RESUMO
ï¡-Thalassemia (AT) is one of the most commonly occurring inherited hematological diseases. However, few treatments are available, and allogeneic bone marrow transplantation (BMT) is the only available therapeutic option for patients with severe AT. Research into AT has remained limited due to a lack of adult mouse models, with severe AT typically resulting in in utero lethality. By using a lipid nanoparticle (LNP) targeting the receptor CD117 and delivering a Cre mRNA (mRNACreLNPCD117), we were able to delete floxed ï¡-globin genes at high efficiency in hematopoietic stem cells (HSC) ex vivo. These cells were then engrafted in the absence or presence of a novel α-globin expressing lentiviral vector (ALS20ï¡I). Myeloablated mice transplanted with mRNACreLNPCD117-treated HSC showed a complete knockout of ï¡-globin genes. They demonstrated a phenotype characterized by the synthesis of hemoglobin H (ï¢-tetramers, ï¢ï´ or HbH), aberrant erythropoiesis, and abnormal organ morphology, culminating in lethality approximately eight weeks following engraftment. Mice receiving mRNACreLNPCD117-treated HSC with at least one copy of ALS20ï¡I survived long-term with normalization of erythropoiesis, decreased the production of HbH, and ameliorated the abnormal organ morphology. Furthermore, we tested ALS20ï¡I in erythroid progenitors derived from ï¡-globin-KO CD34+ and cells isolated from patients with both deletional and non-deletional HbH disease, demonstrating improvement in ï¡-globin/ï¢-globin mRNA ratio and reduction in the formation of HbH by HPLC. Our results demonstrate the broad applicability of LNP for disease modeling, characterization of a novel severe mouse model of AT, and the efficacy of ALS20ï¡I for treating AT.
RESUMO
RNA therapeutics are an emerging, powerful class of drugs with potential applications in a wide range of disorders. A central challenge in their development is the lack of clear pharmacokinetic (PK)-pharmacodynamic relationship, in part due to the significant delay between the kinetics of RNA delivery and the onset of pharmacologic response. To bridge this gap, we have developed a physiologically based PK/pharmacodynamic model for systemically administered mRNA-containing lipid nanoparticles (LNPs) in mice. This model accounts for the physiologic determinants of mRNA delivery, active targeting in the vasculature, and differential transgene expression based on nanoparticle coating. The model was able to well-characterize the blood and tissue PKs of LNPs, as well as the kinetics of tissue luciferase expression measured by ex vivo activity in organ homogenates and bioluminescence imaging in intact organs. The predictive capabilities of the model were validated using a formulation targeted to intercellular adhesion molecule-1 and the model predicted nanoparticle delivery and luciferase expression within a 2-fold error for all organs. This modeling platform represents an initial strategy that can be expanded upon and utilized to predict the in vivo behavior of RNA-containing LNPs developed for an array of conditions and across species.
RESUMO
Effective delivery of mRNA or small molecule drugs to the brain is a significant challenge in developing treatment for acute ischemic stroke (AIS). To address the problem, we have developed targeted nanomedicine to increase drug concentrations in endothelial cells of the blood-brain barrier (BBB) of the injured brain. Inflammation during ischemic stroke causes continuous neuronal death and an increase in the infarct volume. To enable targeted delivery to the inflamed BBB, we conjugated lipid nanocarriers (NCs) with antibodies that bind cell adhesion molecules expressed at the BBB. In the transient middle cerebral artery occlusion mouse model, NCs targeted to vascular cellular adhesion molecule-1 (VCAM) achieved the highest level of brain delivery, nearly two orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles with luciferase-encoding mRNA and Cre-recombinase showed selective expression in the ischemic brain. Anti-inflammatory drugs administered intravenously after ischemic stroke reduced cerebral infarct volume by 62% (interleukin-10 mRNA) or 35% (dexamethasone) only when they were encapsulated in VCAM-targeted NCs. Thus, VCAM-targeted lipid NCs represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS.
Assuntos
Barreira Hematoencefálica , Modelos Animais de Doenças , AVC Isquêmico , Lipossomos , Nanopartículas , Molécula 1 de Adesão de Célula Vascular , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Animais , Camundongos , Molécula 1 de Adesão de Célula Vascular/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Nanopartículas/química , AVC Isquêmico/metabolismo , AVC Isquêmico/tratamento farmacológico , Lipídeos/química , Sistemas de Liberação de Medicamentos/métodos , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , HumanosRESUMO
After more than 100 failed drug trials for acute ischemic stroke (AIS), one of the most commonly cited reasons for the failure has been that drugs achieve very low concentrations in the at-risk penumbra. To address this problem, here we employ nanotechnology to significantly enhance drug concentration in the penumbra's blood-brain barrier (BBB), whose increased permeability in AIS has long been hypothesized to kill neurons by exposing them to toxic plasma proteins. To devise drug-loaded nanocarriers targeted to the BBB, we conjugated them with antibodies that bind to various cell adhesion molecules on the BBB endothelium. In the transient middle cerebral artery occlusion (tMCAO) mouse model, nanocarriers targeted with VCAM antibodies achieved the highest level of brain delivery, nearly 2 orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles loaded with either a small molecule drug (dexamethasone) or mRNA (encoding IL-10) reduced cerebral infarct volume by 35% or 73%, respectively, and both significantly lowered mortality rates. In contrast, the drugs delivered without the nanocarriers had no effect on AIS outcomes. Thus, VCAM-targeted lipid nanoparticles represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS. Graphical abstract: Acute ischemic stroke induces upregulation of VCAM. We specifically targeted upregulated VCAM in the injured region of the brain with drug- or mRNA-loaded targeted nanocarriers. Nanocarriers targeted with VCAM antibodies achieved the highest brain delivery, nearly orders of magnitude higher than untargeted ones. VCAM-targeted nanocarriers loaded with dexamethasone and mRNA encoding IL-10 reduced infarct volume by 35% and 73%, respectively, and improved survival rates.
RESUMO
Hematopoietic stem cells (HSCs) are the source of all blood cells over an individual's lifetime. Diseased HSCs can be replaced with gene-engineered or healthy HSCs through HSC transplantation (HSCT). However, current protocols carry major side effects and have limited access. We developed CD117/LNP-messenger RNA (mRNA), a lipid nanoparticle (LNP) that encapsulates mRNA and is targeted to the stem cell factor receptor (CD117) on HSCs. Delivery of the anti-human CD117/LNP-based editing system yielded near-complete correction of hematopoietic sickle cells. Furthermore, in vivo delivery of pro-apoptotic PUMA (p53 up-regulated modulator of apoptosis) mRNA with CD117/LNP affected HSC function and permitted nongenotoxic conditioning for HSCT. The ability to target HSCs in vivo offers a nongenotoxic conditioning regimen for HSCT, and this platform could be the basis of in vivo genome editing to cure genetic disorders, which would abrogate the need for HSCT.
Assuntos
Edição de Genes , Células-Tronco Hematopoéticas , Proteínas Proto-Oncogênicas c-kit , RNA Mensageiro , Edição de Genes/métodos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , RNA Mensageiro/genética , Animais , Humanos , CamundongosRESUMO
Fibrosis affects millions of people with cardiac disease. We developed a therapeutic approach to generate transient antifibrotic chimeric antigen receptor (CAR) T cells in vivo by delivering modified messenger RNA (mRNA) in T celltargeted lipid nanoparticles (LNPs). The efficacy of these in vivoreprogrammed CAR T cells was evaluated by injecting CD5-targeted LNPs into a mouse model of heart failure. Efficient delivery of modified mRNA encoding the CAR to T lymphocytes was observed, which produced transient, effective CAR T cells in vivo. Antifibrotic CAR T cells exhibited trogocytosis and retained the target antigen as they accumulated in the spleen. Treatment with modified mRNA-targeted LNPs reduced fibrosis and restored cardiac function after injury. In vivo generation of CAR T cells may hold promise as a therapeutic platform to treat various diseases.
Assuntos
Engenharia Celular , Endopeptidases/imunologia , Cardiopatias/terapia , Imunoterapia Adotiva , Lipossomos , Proteínas de Membrana/imunologia , Nanopartículas , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Transferência Adotiva , Animais , Antígenos CD5/imunologia , Endopeptidases/metabolismo , Fibroblastos/imunologia , Fibroblastos/patologia , Fibrose/terapia , Células HEK293 , Cardiopatias/patologia , Insuficiência Cardíaca/terapia , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/patologia , RNA Mensageiro/genética , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Baço/imunologia , TrogocitoseRESUMO
Adjuvants are critical for improving the quality and magnitude of adaptive immune responses to vaccination. Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA vaccines have shown great efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanism of action of this vaccine platform is not well-characterized. Using influenza virus and SARS-CoV-2 mRNA and protein subunit vaccines, we demonstrated that our LNP formulation has intrinsic adjuvant activity that promotes induction of strong T follicular helper cell, germinal center B cell, long-lived plasma cell, and memory B cell responses that are associated with durable and protective antibodies in mice. Comparative experiments demonstrated that this LNP formulation outperformed a widely used MF59-like adjuvant, AddaVax. The adjuvant activity of the LNP relies on the ionizable lipid component and on IL-6 cytokine induction but not on MyD88- or MAVS-dependent sensing of LNPs. Our study identified LNPs as a versatile adjuvant that enhances the efficacy of traditional and next-generation vaccine platforms.
Assuntos
Linfócitos B/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/imunologia , Centro Germinativo/imunologia , SARS-CoV-2/fisiologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinas de mRNA/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adjuvantes Imunológicos , Animais , Células HEK293 , Humanos , Imunidade Humoral , Interleucina-6/genética , Interleucina-6/metabolismo , Lipossomos/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Subunidades Proteicas/genética , Vacinas de mRNA/genéticaRESUMO
Lack or dysfunction of the lymphatics leads to secondary lymphedema formation that seriously reduces the function of the affected organs and results in degradation of quality of life. Currently, there is no definitive treatment option for lymphedema. Here, we utilized nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs) encoding murine Vascular Endothelial Growth Factor C (VEGFC) to stimulate lymphatic growth and function and reduce experimental lymphedema in mouse models. We demonstrated that administration of a single low-dose of VEGFC mRNA-LNPs induced durable, organ-specific lymphatic growth and formation of a functional lymphatic network. Importantly, VEGFC mRNA-LNP treatment reversed experimental lymphedema by restoring lymphatic function without inducing any obvious adverse events. Collectively, we present a novel application of the nucleoside-modified mRNA-LNP platform, describe a model for identifying the organ-specific physiological and pathophysiological roles of the lymphatics, and propose an efficient and safe treatment option that may serve as a novel therapeutic tool to reduce lymphedema.
Assuntos
Linfangiogênese/genética , Vasos Linfáticos/patologia , Linfedema/patologia , Nucleosídeos/metabolismo , Fator C de Crescimento do Endotélio Vascular/genética , Animais , Vasos Sanguíneos/patologia , Proliferação de Células/efeitos dos fármacos , Toxina Diftérica/farmacologia , Modelos Animais de Doenças , Células HEK293 , Humanos , Imunidade/efeitos dos fármacos , Injeções Intradérmicas , Lipídeos/administração & dosagem , Lipídeos/química , Vasos Linfáticos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Nanopartículas/administração & dosagem , Nanopartículas/química , Especificidade de Órgãos , Poli C/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tamoxifeno/farmacologia , Fator C de Crescimento do Endotélio Vascular/administração & dosagem , Fator C de Crescimento do Endotélio Vascular/metabolismoRESUMO
Nucleoside-modified messenger RNA (mRNA)-lipid nanoparticles (LNPs) are the basis for the first two EUA (Emergency Use Authorization) COVID-19 vaccines. The use of nucleoside-modified mRNA as a pharmacological agent opens immense opportunities for therapeutic, prophylactic and diagnostic molecular interventions. In particular, mRNA-based drugs may specifically modulate immune cells, such as T lymphocytes, for immunotherapy of oncologic, infectious and other conditions. The key challenge, however, is that T cells are notoriously resistant to transfection by exogenous mRNA. Here, we report that conjugating CD4 antibody to LNPs enables specific targeting and mRNA interventions to CD4+ cells, including T cells. After systemic injection in mice, CD4-targeted radiolabeled mRNA-LNPs accumulated in spleen, providing â¼30-fold higher signal of reporter mRNA in T cells isolated from spleen as compared with non-targeted mRNA-LNPs. Intravenous injection of CD4-targeted LNPs loaded with Cre recombinase-encoding mRNA provided specific dose-dependent loxP-mediated genetic recombination, resulting in reporter gene expression in about 60% and 40% of CD4+ T cells in spleen and lymph nodes, respectively. T cell phenotyping showed uniform transfection of T cell subpopulations, with no variability in uptake of CD4-targeted mRNA-LNPs in naive, central memory, and effector cells. The specific and efficient targeting and transfection of mRNA to T cells established in this study provides a platform technology for immunotherapy of devastating conditions and HIV cure.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Lipídeos/genética , Lipídeos/imunologia , Nanopartículas/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Recombinação Genética/genética , Animais , COVID-19/imunologia , Vacinas contra COVID-19/imunologia , Humanos , Imunoterapia/métodos , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Recombinação Genética/imunologia , SARS-CoV-2/imunologia , Baço/imunologia , Transfecção/métodosRESUMO
Influenza viruses are respiratory pathogens of public health concern worldwide with up to 650,000 deaths occurring each year. Seasonal influenza virus vaccines are employed to prevent disease, but with limited effectiveness. Development of a universal influenza virus vaccine with the potential to elicit long-lasting, broadly cross-reactive immune responses is necessary for reducing influenza virus prevalence. In this study, we have utilized lipid nanoparticle-encapsulated, nucleoside-modified mRNA vaccines to intradermally deliver a combination of conserved influenza virus antigens (hemagglutinin stalk, neuraminidase, matrix-2 ion channel, and nucleoprotein) and induce strong immune responses with substantial breadth and potency in a murine model. The immunity conferred by nucleoside-modified mRNA-lipid nanoparticle vaccines provided protection from challenge with pandemic H1N1 virus at 500 times the median lethal dose after administration of a single immunization, and the combination vaccine protected from morbidity at a dose of 50 ng per antigen. The broad protective potential of a single dose of combination vaccine was confirmed by challenge with a panel of group 1 influenza A viruses. These findings support the advancement of nucleoside-modified mRNA-lipid nanoparticle vaccines expressing multiple conserved antigens as universal influenza virus vaccine candidates.
Assuntos
Antígenos Virais/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Nucleosídeos/química , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas Sintéticas/administração & dosagem , Animais , Anticorpos Antivirais/metabolismo , Antígenos Virais/química , Modelos Animais de Doenças , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/química , Vacinas contra Influenza/imunologia , Injeções Intradérmicas , Lipossomos , Camundongos , Células NIH 3T3 , Nanopartículas , Neuraminidase/química , Neuraminidase/genética , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/genética , Infecções por Orthomyxoviridae/imunologia , Vacinas Sintéticas/química , Vacinas Sintéticas/imunologia , Vacinas de mRNARESUMO
Malaria caused by Plasmodium falciparum remains the leading single-agent cause of mortality in children1, yet the promise of an effective vaccine has not been fulfilled. Here, using our previously described differential screening method to analyse the proteome of blood-stage P. falciparum parasites2, we identify P. falciparum glutamic-acid-rich protein (PfGARP) as a parasite antigen that is recognized by antibodies in the plasma of children who are relatively resistant-but not those who are susceptible-to malaria caused by P. falciparum. PfGARP is a parasite antigen of 80 kDa that is expressed on the exofacial surface of erythrocytes infected by early-to-late-trophozoite-stage parasites. We demonstrate that antibodies against PfGARP kill trophozoite-infected erythrocytes in culture by inducing programmed cell death in the parasites, and that vaccinating non-human primates with PfGARP partially protects against a challenge with P. falciparum. Furthermore, our longitudinal cohort studies showed that, compared to individuals who had naturally occurring anti-PfGARP antibodies, Tanzanian children without anti-PfGARP antibodies had a 2.5-fold-higher risk of severe malaria and Kenyan adolescents and adults without these antibodies had a twofold-higher parasite density. By killing trophozoite-infected erythrocytes, PfGARP could synergize with other vaccines that target parasite invasion of hepatocytes or the invasion of and egress from erythrocytes.
Assuntos
Apoptose/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Parasitos/imunologia , Plasmodium falciparum/citologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Adolescente , Adulto , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Aotidae/imunologia , Aotidae/parasitologia , Caspases/metabolismo , Criança , Estudos de Coortes , DNA de Protozoário/química , DNA de Protozoário/metabolismo , Ativação Enzimática , Eritrócitos/parasitologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Quênia , Vacinas Antimaláricas/imunologia , Malária Falciparum/parasitologia , Masculino , Camundongos , Parasitos/citologia , Parasitos/crescimento & desenvolvimento , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/química , Tanzânia , Trofozoítos/citologia , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/imunologia , Vacúolos/imunologiaRESUMO
Drug targeting to inflammatory brain pathologies such as stroke and traumatic brain injury remains an elusive goal. Using a mouse model of acute brain inflammation induced by local tumor necrosis factor alpha (TNFα), we found that uptake of intravenously injected antibody to vascular cell adhesion molecule 1 (anti-VCAM) in the inflamed brain is >10-fold greater than antibodies to transferrin receptor-1 and intercellular adhesion molecule 1 (TfR-1 and ICAM-1). Furthermore, uptake of anti-VCAM/liposomes exceeded that of anti-TfR and anti-ICAM counterparts by â¼27- and â¼8-fold, respectively, achieving brain/blood ratio >300-fold higher than that of immunoglobulin G/liposomes. Single-photon emission computed tomography imaging affirmed specific anti-VCAM/liposome targeting to inflamed brain in mice. Intravital microscopy via cranial window and flow cytometry showed that in the inflamed brain anti-VCAM/liposomes bind to endothelium, not to leukocytes. Anti-VCAM/LNP selectively accumulated in the inflamed brain, providing de novo expression of proteins encoded by cargo messenger RNA (mRNA). Anti-VCAM/LNP-mRNA mediated expression of thrombomodulin (a natural endothelial inhibitor of thrombosis, inflammation, and vascular leakage) and alleviated TNFα-induced brain edema. Thus VCAM-directed nanocarriers provide a platform for cerebrovascular targeting to inflamed brain, with the goal of normalizing the integrity of the blood-brain barrier, thus benefiting numerous brain pathologies.
Assuntos
Anticorpos/administração & dosagem , Barreira Hematoencefálica/efeitos dos fármacos , Encefalite/tratamento farmacológico , Endotélio Vascular/efeitos dos fármacos , Nanomedicina/métodos , Animais , Barreira Hematoencefálica/imunologia , Encefalite/genética , Encefalite/imunologia , Endotélio Vascular/imunologia , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Camundongos , Receptores da Transferrina/genética , Receptores da Transferrina/imunologia , Trombomodulina/genética , Trombomodulina/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/imunologiaRESUMO
Maternal antibodies provide short-term protection to infants against many infections. However, they can inhibit de novo antibody responses in infants elicited by infections or vaccination, leading to increased long-term susceptibility to infectious diseases. Thus, there is a need to develop vaccines that are able to elicit protective immune responses in the presence of antigen-specific maternal antibodies. Here, we used a mouse model to demonstrate that influenza virus-specific maternal antibodies inhibited de novo antibody responses in mouse pups elicited by influenza virus infection or administration of conventional influenza vaccines. We found that a recently developed influenza vaccine, nucleoside-modified mRNA encapsulated in lipid nanoparticles (mRNA-LNP), partially overcame this inhibition by maternal antibodies. The mRNA-LNP influenza vaccine established long-lived germinal centers in the mouse pups and elicited stronger antibody responses than did a conventional influenza vaccine approved for use in humans. Vaccination with mRNA-LNP vaccines may offer a promising strategy for generating robust immune responses in infants in the presence of maternal antibodies.
Assuntos
Formação de Anticorpos/imunologia , Nucleosídeos/metabolismo , RNA Mensageiro/imunologia , Vacinação , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos Antivirais/imunologia , Modelos Animais de Doenças , Feminino , Centro Germinativo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vacinas contra Influenza/imunologia , Lipídeos/química , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nanopartículas/química , Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologiaAssuntos
Polineuropatias/tratamento farmacológico , RNA Interferente Pequeno/uso terapêutico , Animais , Aprovação de Drogas , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Desenvolvimento de Medicamentos , Humanos , Nanomedicina , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , Pesquisa Translacional BiomédicaRESUMO
In this work, we studied the expression kinetics and innate immune response of a self-amplifying mRNA (sa-RNA) after electroporation and lipid-nanoparticle (LNP)-mediated delivery in the skin of mice. Intradermal electroporation of the sa-RNA resulted in a plateau-shaped expression, with the plateau between day 3 and day 10. The overall protein expression of sa-RNA was significantly higher than that obtained after electroporation of plasmid DNA (pDNA) or non-replication mRNAs. Moreover, using IFN-ß reporter mice, we elucidated that intradermal electroporation of sa-RNA induced a short-lived moderate innate immune response, which did not affect the expression of the sa-RNA. A completely different expression profile and innate immune response were observed when LNPs were used. The expression peaked 24 h after intradermal injection of sa-RNA-LNPs and subsequently showed a sharp drop. This drop might be explained by a translational blockage caused by the strong innate immune response that we observed in IFN-ß reporter mice shortly (4 h) after intradermal injection of sa-RNA-LNPs. A final interesting observation was the capacity of sa-RNA-LNPs to transfect the draining lymph nodes after intradermal injection.
RESUMO
Despite the enormous effort in the development of effective vaccines against HIV-1, no vaccine candidate has elicited broadly neutralizing antibodies in humans. Thus, generation of more effective anti-HIV vaccines is critically needed. Here we characterize the immune responses induced by nucleoside-modified and purified mRNA-lipid nanoparticle (mRNA-LNP) vaccines encoding the clade C transmitted/founder HIV-1 envelope (Env) 1086C. Intradermal vaccination with nucleoside-modified 1086C Env mRNA-LNPs elicited high levels of gp120-specific antibodies in rabbits and rhesus macaques. Antibodies generated in rabbits neutralized a tier 1 virus, but no tier 2 neutralization activity could be measured. Importantly, three of six non-human primates developed antibodies that neutralized the autologous tier 2 strain. Despite stable anti-gp120 immunoglobulin G (IgG) levels, tier 2 neutralization titers started to drop 4 weeks after booster immunizations. Serum from both immunized rabbits and non-human primates demonstrated antibody-dependent cellular cytotoxicity activity. Collectively, these results are supportive of continued development of nucleoside-modified and purified mRNA-LNP vaccines for HIV. Optimization of Env immunogens and vaccination protocols are needed to increase antibody neutralization breadth and durability.
RESUMO
It has previously been shown that engineered zinc finger nucleases (ZFNs) can be packaged into adeno-associated viruses (AAVs) and delivered intravenously into mice, non-human primates, and most recently, humans to induce highly efficient therapeutic genome editing in the liver. Lipid nanoparticles (LNPs) are synthetic delivery vehicles that enable repeat administration and are not limited by the presence of preexisting neutralizing antibodies in patients. Here, we show that mRNA encoding ZFNs formulated into LNP can enable >90% knockout of gene expression in mice by targeting the TTR or PCSK9 gene, at mRNA doses 10-fold lower than has ever been reported. Additionally, co-delivering mRNA-LNP containing ZFNs targeted to intron 1 of the ALB locus with AAV packaged with a promoterless human IDS or FIX therapeutic transgene can result in high levels of targeted integration and subsequent therapeutically relevant levels of protein expression in mice. Finally, we show repeat administration of ZFN mRNA-LNP after a single AAV donor dose results in significantly increased levels of genome editing and transgene expression compared to a single dose. These results demonstrate LNP-mediated ZFN mRNA delivery can drive highly efficient levels of in vivo genome editing and can potentially offer a new treatment modality for a variety of diseases.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Edição de Genes/métodos , Nanopartículas/administração & dosagem , RNA Mensageiro/administração & dosagem , Nucleases de Dedos de Zinco/administração & dosagem , Animais , Células Cultivadas , Dependovirus/genética , Feminino , Técnicas de Inativação de Genes , Vetores Genéticos , Hepatócitos/metabolismo , Íntrons/genética , Lipídeos/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pré-Albumina/genética , Pró-Proteína Convertase 9/genética , RNA Mensageiro/genética , Transgenes/genética , Nucleases de Dedos de Zinco/farmacologiaRESUMO
Systemic administration of lipid nanoparticle (LNP)-encapsulated messenger RNA (mRNA) leads predominantly to hepatic uptake and expression. Here, we conjugated nucleoside-modified mRNA-LNPs with antibodies (Abs) specific to vascular cell adhesion molecule, PECAM-1. Systemic (intravenous) administration of Ab/LNP-mRNAs resulted in profound inhibition of hepatic uptake concomitantly with ~200-fold and 25-fold elevation of mRNA delivery and protein expression in the lungs compared to non-targeted counterparts. Unlike hepatic delivery of LNP-mRNA, Ab/LNP-mRNA is independent of apolipoprotein E. Vascular re-targeting of mRNA represents a promising, powerful, and unique approach for novel experimental and clinical interventions in organs of interest other than liver.
Assuntos
Apolipoproteínas E/metabolismo , Sistemas de Liberação de Medicamentos , Endotélio Vascular/metabolismo , Nanopartículas/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , RNA Mensageiro/administração & dosagem , Administração Intravenosa , Animais , Linhagem Celular , Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Células Endoteliais da Veia Umbilical Humana , Humanos , Imunoconjugados/metabolismo , Camundongos Endogâmicos C57BL , RNA Mensageiro/farmacocinética , Distribuição TecidualRESUMO
Currently available influenza virus vaccines have inadequate effectiveness and are reformulated annually due to viral antigenic drift. Thus, development of a vaccine that confers long-term protective immunity against antigenically distant influenza virus strains is urgently needed. The highly conserved influenza virus hemagglutinin (HA) stalk represents one of the potential targets of broadly protective/universal influenza virus vaccines. Here, we evaluate a potent broadly protective influenza virus vaccine candidate that uses nucleoside-modified and purified mRNA encoding full-length influenza virus HA formulated in lipid nanoparticles (LNPs). We demonstrate that immunization with HA mRNA-LNPs induces antibody responses against the HA stalk domain of influenza virus in mice, rabbits, and ferrets. The HA stalk-specific antibody response is associated with protection from homologous, heterologous, and heterosubtypic influenza virus infection in mice.