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1.
Proc Natl Acad Sci U S A ; 121(2): e2308415120, 2024 Jan 09.
Artículo en Inglés | MEDLINE | ID: mdl-38150477

RESUMEN

Genomic DNA of the cyanophage S-2L virus is composed of 2-aminoadenine (Z), thymine (T), guanine (G), and cytosine (C), forming the genetic alphabet ZTGC, which violates Watson-Crick base pairing rules. The Z-base has an extra amino group on the two position that allows the formation of a third hydrogen bond with thymine in DNA strands. Here, we explored and expanded applications of this non-Watson-Crick base pairing in protein expression and gene editing. Both ZTGC-DNA (Z-DNA) and ZUGC-RNA (Z-RNA) produced in vitro show detectable compatibility and can be decoded in mammalian cells, including Homo sapiens cells. Z-crRNA can guide CRISPR-effectors SpCas9 and LbCas12a to cleave specific DNA through non-Watson-Crick base pairing and boost cleavage activities compared to A-crRNA. Z-crRNA can also allow for efficient gene and base editing in human cells. Together, our results help pave the way for potential strategies for optimizing DNA or RNA payloads for gene editing therapeutics and give insights to understanding the natural Z-DNA genome.


Asunto(s)
Emparejamiento Base , Sistemas CRISPR-Cas , ADN de Forma Z , Edición Génica , Humanos , ADN/genética , ADN/química , ADN de Forma Z/genética , Edición Génica/métodos , ARN/genética , ARN Guía de Sistemas CRISPR-Cas , Timina/química
2.
Trends Genet ; 39(3): 208-216, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36669950

RESUMEN

There is wide interest in applying genome-editing tools to prevent, treat, and cure a variety of diseases. Since the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, these techniques have been used in combination with different delivery systems to create highly efficacious treatment options. Each delivery system has its own advantages and disadvantages and is being used for various applications. With the large number of gene-editing applications being studied but very few being brought into the clinic, we review current progress in the field, specifically where genome editing has been applied in vivo and in the clinic, and identify current challenges and areas of future growth.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Edición Génica/métodos , Resultado del Tratamiento
3.
Proc Natl Acad Sci U S A ; 120(52): e2311752120, 2023 Dec 26.
Artículo en Inglés | MEDLINE | ID: mdl-38134199

RESUMEN

The emergence of highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that are resistant to the current COVID-19 vaccines highlights the need for continued development of broadly protective vaccines for the future. Here, we developed two messenger RNA (mRNA)-lipid nanoparticle (LNP) vaccines, TU88mCSA and ALCmCSA, using the ancestral SARS-CoV-2 spike sequence, optimized 5' and 3' untranslated regions (UTRs), and LNP combinations. Our data showed that these nanocomplexes effectively activate CD4+ and CD8+ T cell responses and humoral immune response and provide complete protection against WA1/2020, Omicron BA.1 and BQ.1 infection in hamsters. Critically, in Omicron BQ.1 challenge hamster models, TU88mCSA and ALCmCSA not only induced robust control of virus load in the lungs but also enhanced protective efficacy in the upper respiratory airways. Antigen-specific immune analysis in mice revealed that the observed cross-protection is associated with superior UTRs [Carboxylesterase 1d (Ces1d)/adaptor protein-3ß (AP3B1)] and LNP formulations that elicit robust lung tissue-resident memory T cells. Strong protective effects of TU88mCSA or ALCmCSA against both WA1/2020 and VOCs suggest that this mRNA-LNP combination can be a broadly protective vaccine platform in which mRNA cargo uses the ancestral antigen sequence regardless of the antigenic drift. This approach could be rapidly adapted for clinical use and timely deployment of vaccines against emerging and reemerging VOCs.


Asunto(s)
Vacunas contra la COVID-19 , COVID-19 , Cricetinae , Animales , Humanos , Ratones , ARN Mensajero/genética , Vacunas contra la COVID-19/genética , Vacunas de ARNm , SARS-CoV-2/genética , COVID-19/prevención & control , Regiones no Traducidas 3' , Anticuerpos Neutralizantes , Anticuerpos Antivirales
4.
Proc Natl Acad Sci U S A ; 119(34): e2207841119, 2022 08 23.
Artículo en Inglés | MEDLINE | ID: mdl-35969778

RESUMEN

The targeted delivery of messenger RNA (mRNA) to desired organs remains a great challenge for in vivo applications of mRNA technology. For mRNA vaccines, the targeted delivery to the lymph node (LN) is predicted to reduce side effects and increase the immune response. In this study, we explored an endogenously LN-targeting lipid nanoparticle (LNP) without the modification of any active targeting ligands for developing an mRNA cancer vaccine. The LNP named 113-O12B showed increased and specific expression in the LN compared with LNP formulated with ALC-0315, a synthetic lipid used in the COVID-19 vaccine Comirnaty. The targeted delivery of mRNA to the LN increased the CD8+ T cell response to the encoded full-length ovalbumin (OVA) model antigen. As a result, the protective and therapeutic effect of the OVA-encoding mRNA vaccine on the OVA-antigen-bearing B16F10 melanoma model was also improved. Moreover, 113-O12B encapsulated with TRP-2 peptide (TRP2180-188)-encoding mRNA also exhibited excellent tumor inhibition, with the complete response of 40% in the regular B16F10 tumor model when combined with anti-programmed death-1 (PD-1) therapy, revealing broad application of 113-O12B from protein to peptide antigens. All the treated mice showed long-term immune memory, hindering the occurrence of tumor metastatic nodules in the lung in the rechallenging experiments that followed. The enhanced antitumor efficacy of the LN-targeting LNP system shows great potential as a universal platform for the next generation of mRNA vaccines.


Asunto(s)
Vacunas contra el Cáncer , Nanopartículas , Neoplasias , Vacunas de ARNm , Amino Alcoholes , Animales , Antígenos/metabolismo , Linfocitos T CD8-positivos , Vacunas contra el Cáncer/uso terapéutico , Decanoatos , Memoria Inmunológica , Liposomas , Ganglios Linfáticos , Ratones , Metástasis de la Neoplasia/prevención & control , Neoplasias/terapia , Ovalbúmina , Vacunas de ARNm/uso terapéutico
5.
Proc Natl Acad Sci U S A ; 119(8)2022 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-35173043

RESUMEN

Safe and efficacious systemic delivery of messenger RNA (mRNA) to specific organs and cells in vivo remains the major challenge in the development of mRNA-based therapeutics. Targeting of systemically administered lipid nanoparticles (LNPs) coformulated with mRNA has largely been confined to the liver and spleen. Using a library screening approach, we identified that N-series LNPs (containing an amide bond in the tail) are capable of selectively delivering mRNA to the mouse lung, in contrast to our previous discovery that O-series LNPs (containing an ester bond in the tail) that tend to deliver mRNA to the liver. We analyzed the protein corona on the liver- and lung-targeted LNPs using liquid chromatography-mass spectrometry and identified a group of unique plasma proteins specifically absorbed onto the surface that may contribute to the targetability of these LNPs. Different pulmonary cell types can also be targeted by simply tuning the headgroup structure of N-series LNPs. Importantly, we demonstrate here the success of LNP-based RNA therapy in a preclinical model of lymphangioleiomyomatosis (LAM), a destructive lung disease caused by loss-of-function mutations in the Tsc2 gene. Our lung-targeting LNP exhibited highly efficient delivery of the mouse tuberous sclerosis complex 2 (Tsc2) mRNA for the restoration of TSC2 tumor suppressor in tumor and achieved remarkable therapeutic effect in reducing tumor burden. This research establishes mRNA LNPs as a promising therapeutic intervention for the treatment of LAM.


Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Linfangioleiomiomatosis/tratamiento farmacológico , ARN Mensajero/administración & dosificación , Animales , Femenino , Técnicas de Transferencia de Gen , Ingeniería Genética/métodos , Liposomas/química , Liposomas/farmacología , Pulmón/citología , Pulmón/patología , Enfermedades Pulmonares/tratamiento farmacológico , Enfermedades Pulmonares/metabolismo , Linfangioleiomiomatosis/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Nanopartículas/química , Corona de Proteínas/química , Corona de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/farmacología , ARN Interferente Pequeño/metabolismo
6.
Biochemistry ; 2024 Feb 09.
Artículo en Inglés | MEDLINE | ID: mdl-38334719

RESUMEN

Protein therapeutics are an expanding area for research and drug development, and lipid nanoparticles (LNPs) are the most prominent nonviral vehicles for protein delivery. The most common methods for assessing protein delivery by LNPs include assays that measure the total amount of protein taken up by cells and assays that measure the phenotypic changes associated with protein delivery. However, assays for total cellular uptake include large amounts of protein that are trapped in endosomes or are otherwise nonfunctional. Assays for functional delivery are important, but the readouts are indirect and amplified, limiting the quantitative interpretation. Here, we apply an assay for cytosolic delivery, the chloroalkane penetration assay (CAPA), to measure the cytosolic delivery of a (-30) green fluorescent protein (GFP) fused to Cre recombinase (Cre(-30)GFP) fusion protein by LNPs. We compare these data to the data from total cellular uptake and functional delivery assays to provide a richer analysis of uptake and endosomal escape for LNP-mediated protein delivery. We also use CAPA for a screen of a small library of lipidoids, identifying those with a promising ability to deliver Cre(-30)GFP to the cytosol of mammalian cells. With careful controls and optimized conditions, we expect that CAPA will be a useful tool for investigating the rate, efficiency, and mechanisms of LNP-mediated delivery of therapeutic proteins.

7.
Nature ; 553(7687): 217-221, 2018 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-29258297

RESUMEN

Although genetic factors contribute to almost half of all cases of deafness, treatment options for genetic deafness are limited. We developed a genome-editing approach to target a dominantly inherited form of genetic deafness. Here we show that cationic lipid-mediated in vivo delivery of Cas9-guide RNA complexes can ameliorate hearing loss in a mouse model of human genetic deafness. We designed and validated, both in vitro and in primary fibroblasts, genome editing agents that preferentially disrupt the dominant deafness-associated allele in the Tmc1 (transmembrane channel-like gene family 1) Beethoven (Bth) mouse model, even though the mutant Tmc1Bth allele differs from the wild-type allele at only a single base pair. Injection of Cas9-guide RNA-lipid complexes targeting the Tmc1Bth allele into the cochlea of neonatal Tmc1Bth/+ mice substantially reduced progressive hearing loss. We observed higher hair cell survival rates and lower auditory brainstem response thresholds in injected ears than in uninjected ears or ears injected with control complexes that targeted an unrelated gene. Enhanced acoustic startle responses were observed among injected compared to uninjected Tmc1Bth/+ mice. These findings suggest that protein-RNA complex delivery of target gene-disrupting agents in vivo is a potential strategy for the treatment of some types of autosomal-dominant hearing loss.


Asunto(s)
Proteínas Asociadas a CRISPR/administración & dosificación , Edición Génica/métodos , Genes Dominantes/genética , Terapia Genética/métodos , Pérdida Auditiva/genética , Estimulación Acústica , Alelos , Animales , Animales Recién Nacidos , Umbral Auditivo , Secuencia de Bases , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/uso terapéutico , Sistemas CRISPR-Cas , Supervivencia Celular , Cóclea/citología , Cóclea/metabolismo , Modelos Animales de Enfermedad , Potenciales Evocados Auditivos del Tronco Encefálico , Femenino , Fibroblastos , Células Ciliadas Auditivas/citología , Pérdida Auditiva/fisiopatología , Pérdida Auditiva/prevención & control , Humanos , Liposomas , Masculino , Proteínas de la Membrana/genética , Ratones , Reflejo de Sobresalto
8.
Proc Natl Acad Sci U S A ; 118(10)2021 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-33649229

RESUMEN

Loss-of-function mutations in Angiopoietin-like 3 (Angptl3) are associated with lowered blood lipid levels, making Angptl3 an attractive therapeutic target for the treatment of human lipoprotein metabolism disorders. In this study, we developed a lipid nanoparticle delivery platform carrying Cas9 messenger RNA (mRNA) and guide RNA for CRISPR-Cas9-based genome editing of Angptl3 in vivo. This system mediated specific and efficient Angptl3 gene knockdown in the liver of wild-type C57BL/6 mice, resulting in profound reductions in serum ANGPTL3 protein, low density lipoprotein cholesterol, and triglyceride levels. Our delivery platform is significantly more efficient than the FDA-approved MC-3 LNP, the current gold standard. No evidence of off-target mutagenesis was detected at any of the nine top-predicted sites, and no evidence of toxicity was detected in the liver. Importantly, the therapeutic effect of genome editing was stable for at least 100 d after a single dose administration. This study highlights the potential of LNP-mediated delivery as a specific, effective, and safe platform for Cas9-based therapeutics.


Asunto(s)
Proteínas Similares a la Angiopoyetina , Proteína 9 Asociada a CRISPR/genética , Portadores de Fármacos , Edición Génica , Lípidos , Hígado/metabolismo , Nanopartículas/química , ARN Guía de Kinetoplastida , ARN Mensajero , Proteína 3 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina/genética , Proteínas Similares a la Angiopoyetina/metabolismo , Animales , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacología , Femenino , Lípidos/química , Lípidos/farmacocinética , Lípidos/farmacología , Ratones , Ratones Endogámicos BALB C , Especificidad de Órganos , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/farmacocinética , ARN Guía de Kinetoplastida/farmacología , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/farmacocinética , ARN Mensajero/farmacología
9.
J Am Chem Soc ; 145(1): 551-559, 2023 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-36537880

RESUMEN

Photoresponsive inhibitor and noninhibitor systems have been developed to achieve on-demand enzyme activity control. However, inhibitors are only effective for a specific and narrow range of enzymes. Noninhibitor systems usually require mutation and modification of the enzymes, leading to irreversible loss of enzymatic activities. Inspired by biological membranes, we herein report a lipidoid-based artificial compartment composed of azobenzene (Azo) lipidoids and helper lipids, which can bidirectionally regulate the activity of the encapsulated enzymes by light. In this system, the reversible photoisomerization of Azo lipidoids triggered by UV/vis light creates a continuous rotation-inversion movement, thereby enhancing the permeability of the compartment membrane and allowing substrates to pass through. Moreover, the membrane can revert to its impermeable state when light is removed. Thus, enzyme activity can be switched on and off when encapsulating enzymes in the compartments. Importantly, since neither mutation nor modification is required, negligible loss of activity is observed for the encapsulated enzymes after repeated activation and inhibition. Furthermore, this approach provides a generic strategy for controlling multiple enzymes by forgoing the use of inhibitors and may broaden the applications of enzymes in biological mechanism research and precision medicine.


Asunto(s)
Compuestos Azo , Rayos Ultravioleta , Membrana Celular , Compuestos Azo/farmacología
10.
Annu Rev Biomed Eng ; 24: 85-109, 2022 06 06.
Artículo en Inglés | MEDLINE | ID: mdl-35231177

RESUMEN

mRNA vaccines have brought about a great revolution in the vaccine fields owing to their simplicity and adaptability in antigen design, potential to induce both humoral and cell-mediated immune responses and demonstrated high efficacy, and rapid and low-cost production by using the same manufacturing platform for different mRNA vaccines. Multiple mRNA vaccines have been investigated for both infectious diseases and cancers, showing significant superiority to other types of vaccines. Although great success of mRNA vaccines has been achieved in the control of the coronavirus disease 2019 pandemic, there are still multiple challenges for the future development of mRNA vaccines. In this review, the most recent developments of mRNA vaccines against both infectious diseases and cancers are summarized for an overview of this field. Moreover, the challenges are also discussed on the basis of these developments.


Asunto(s)
COVID-19 , Enfermedades Transmisibles , Neoplasias , COVID-19/prevención & control , Humanos , ARN Mensajero/genética , Vacunas Sintéticas , Vacunas de ARNm
11.
Acc Chem Res ; 54(21): 4001-4011, 2021 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-34668716

RESUMEN

Since the U.S. Food and Drug Administration (FDA) granted emergency use authorization for two mRNA vaccines against SARS-CoV-2, mRNA-based technology has attracted broad attention from the scientific community to investors. When delivered intracellularly, mRNA has the ability to produce various therapeutic proteins, enabling the treatment of a variety of illnesses, including but not limited to infectious diseases, cancers, and genetic diseases. Accordingly, mRNA holds significant therapeutic potential and provides a promising means to target historically hard-to-treat diseases. Current clinical efforts harnessing mRNA-based technology are focused on vaccination, cancer immunotherapy, protein replacement therapy, and genome editing. The clinical translation of mRNA-based technology has been made possible by leveraging nanoparticle delivery methods. However, the application of mRNA for therapeutic purposes is still challenged by the need for specific, efficient, and safe delivery systems.This Account highlights key advances in designing and developing combinatorial synthetic lipid nanoparticles (LNPs) with distinct chemical structures and properties for in vitro and in vivo intracellular mRNA delivery. LNPs represent the most advanced nonviral nanoparticle delivery systems that have been extensively investigated for nucleic acid delivery. The aforementioned COVID-19 mRNA vaccines and one LNP-based small interfering RNA (siRNA) drug (ONPATTRO) have received clinical approval from the FDA, highlighting the success of synthetic ionizable lipids for in vivo nucleic acid delivery. In this Account, we first summarize the research efforts from our group on the development of bioreducible and biodegradable LNPs by leveraging the combinatorial chemistry strategy, such as the Michael addition reaction, which allows us to easily generate a large set of lipidoids with diverse chemical structures. Next, we discuss the utilization of a library screening strategy to identify optimal LNPs for targeted mRNA delivery and showcase the applications of the optimized LNPs in cell engineering and genome editing. Finally, we outline key challenges to the clinical translation of mRNA-based therapies and propose an outlook for future directions of the chemical design and optimization of LNPs to improve the safety and specificity of mRNA drugs. We hope this Account provides insight into the rational design of LNPs for facilitating the development of mRNA therapeutics, a transformative technology that promises to revolutionize future medicine.


Asunto(s)
Vacunas contra la COVID-19/farmacología , Edición Génica , Técnicas de Transferencia de Gen , Lípidos/química , Nanopartículas/química , ARN Mensajero/farmacología , Vacunas contra la COVID-19/química , Terapia Genética , Humanos , ARN Mensajero/química , SARS-CoV-2/efectos de los fármacos , Tratamiento Farmacológico de COVID-19
12.
Langmuir ; 38(30): 9092-9098, 2022 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-35852946

RESUMEN

The field of nanomedicine has rapidly grown in the past decades. Although a few nanomedicines are available in the market for clinical use, it is still challenging to develop nanomedicine targeting tissues beyond the liver. It has been recognized that even though the nanoparticles are modified with targeting ligands, the formation of a protein corona on the surface of nanoparticles in the biological fluids results in limited progress in nanoparticle-based drug delivery to specific cells or tissues. In this Perspective, we will discuss the role of surface properties in determining the formation of the protein corona and summarize the recent progress in engineering the nano/bio interface for protein-corona-mediated cell- and organ-selective drug delivery. Moreover, current challenges in the field and insights into designing new strategies for targeting drug delivery with a better understanding of the protein corona will be discussed.


Asunto(s)
Nanopartículas , Corona de Proteínas , Sistemas de Liberación de Medicamentos , Ingeniería , Nanomedicina/métodos
13.
Bioconjug Chem ; 31(7): 1835-1843, 2020 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-32520527

RESUMEN

Lipidoid nanoparticles have been demonstrated to be effective for intracellular delivery of small molecule drugs, proteins, and nucleic acids. Stimuli-responsive lipidoid nanoparticles are able to further improve delivery efficacy and reduce carrier-induced toxicity. Our group previously developed reduction and photoresponsive combinatorial libraries of lipidoid nanoparticles for small molecule and biologics delivery. Herein, we describe the synthesis, characterization, and intracellular mRNA delivery application of a new library of pH-responsive lipidoid nanoparticles. The acid-degradable cyclic benzylidene acetal-containing cationic lipidoids (R-O16CBA) were synthesized through a multistep reaction and characterized by NMR and MS. The acid-triggered degradation of lipidoids was studied using NMR, MS, DLS, and TEM. The results revealed that the R-O16CBA lipidoid can be completely degraded at pH 5. The R-O16CBA lipidoid nanoparticles were then fabricated with different formulations of DOPE and cholesterol and tested in vitro for intracellular mRNA delivery.


Asunto(s)
Acetales/química , Técnicas Químicas Combinatorias , Portadores de Fármacos , Concentración de Iones de Hidrógeno , Lípidos/química , Nanopartículas/química , ARN Mensajero/administración & dosificación , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía Electrónica de Transmisión , Análisis Espectral/métodos
14.
Acc Chem Res ; 52(3): 665-675, 2019 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-30586281

RESUMEN

The use of protein to precisely manipulate cell signaling is an effective approach for controlling cell fate and developing precision medicine. More recently, programmable nucleases, such as CRISPR/Cas9, have shown extremely high potency for editing genetic flow of mammalian cells, and for treating genetic disorders. The therapeutic potential of proteins with an intracellular target, however, is mostly challenged by their low cell impermeability. Therefore, a developing delivery system to transport protein to the site of action in a spatiotemporal controlled manner is of great importance to expand the therapeutic index of the protein. In this Account, we first summarize our most recent advances in designing combinatorial lipid nanoparticles with diverse chemical structures for intracellular protein delivery. By designing parallel Michael addition or ring-opening reaction of aliphatic amines, we have generated a combinatorial library of cationic lipids, and identified several leading nanoparticle formulations for intracellular protein delivery both in vitro and in vivo. Moreover, we optimized the chemical structure of lipids to control lipid degradation and protein release inside cells for CRISPR/Cas9 genome-editing protein delivery. In the second part of this Account, we survey our recent endeavor in developing a chemical approach to modify protein, in particular, coupled with the nanoparticle delivery platform, to improve protein delivery for targeted diseases treatment and genome editing. Chemical modification of protein is a useful tool to modulate protein function and to improve the therapeutic index of protein drugs. Herein, we mostly summarize our recent advances on designing chemical approaches to modify protein with following unique findings: (1) chemically modified protein shows selective turn-on activity based on the specific intracellular microenvironment, with which we were able to protein-based targeted cancer therapy; (2) the conjugation of hyaluronic acid (HA) to protein allows cancer cell surface receptor-targeted delivery of protein; (3) the introduction of nonpeptidic boronic acid into protein enabled cell nucleus targeted delivery; this is the first report that a nonpeptidic signal can direct protein to subcellular compartment; and (4) the fusion of protein with negatively supercharged green fluorescent protein (GFP) facilitates the self-assembly of protein with lipid nanoparticle for genome-editing protein delivery. At the end of the Account, we give a perspective of expanding the chemistry that could be integrated to design biocompatible lipid nanocarriers for protein delivery and genome editing in vitro and in vivo, as well as the chemical approaches that we can harness to modulate protein activity in live cells for targeted diseases treatment.


Asunto(s)
Portadores de Fármacos/química , Lípidos/química , Nanopartículas/química , Proteínas/metabolismo , Animales , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Línea Celular Tumoral , Edición Génica/métodos , Técnicas de Transferencia de Gen , Humanos , Ratones , Ratas
15.
Angew Chem Int Ed Engl ; 59(45): 20083-20089, 2020 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-32662132

RESUMEN

Engineering T lymphocytes is an emerging approach in a variety of biomedical applications. However, delivering large biologics to primary T lymphocytes directly in vivo is technically challenging due to the low transfection efficacy. Herein, we investigated a library of synthetic lipid-like molecules (lipidoids) for their capability of delivering mRNA into primary T lymphocytes both ex vivo and in vivo. We initially screened a library with a large structural variety of lipidoids ex vivo and identified imidazole-containing lipidoids that are particularly potent in T lymphocytes transfection. We further optimized lipidoid structures by constructing and screening a detailed lipidoid library containing imidazole or imidazole analogues to perform a structure-activity correlation analysis. Using the lead lipidoid as a delivery vehicle for Cre mRNA in vivo through intravenous injection, we achieved 8.2 % gene recombination in mouse T lymphocytes.


Asunto(s)
Imidazoles/química , Lípidos/química , ARN Mensajero/administración & dosificación , Linfocitos T/metabolismo , Animales , Técnicas de Transferencia de Gen , Células HeLa , Humanos , Ratones
16.
Angew Chem Int Ed Engl ; 59(35): 14957-14964, 2020 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-32438474

RESUMEN

Developing safe and efficient delivery systems for therapeutic biomacromolecules is a long-standing challenge. Herein, we report a newly developed combinatorial library of cholesteryl-based disulfide bond-containing biodegradable cationic lipidoid nanoparticles. We have identified a subset of this library which is effective for protein and mRNA delivery in vitro and in vivo. These lipidoids showed comparable transfection efficacies but much lower cytotoxicities compared to the Lpf2k in vitro. In vivo studies in adult mice demonstrated the successful delivery of genome engineering protein and mRNA molecules in the skeletal muscle (via intramuscular injection), lung and spleen (via intravenous injection), and brain (via lateral ventricle infusion).


Asunto(s)
Nanopartículas/metabolismo , Proteínas/síntesis química , ARN Mensajero/química , Animales , Humanos , Ratones
17.
Gene Ther ; 26(5): 187-197, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30926963

RESUMEN

Patients with immunoglobulin (Ig) light-chain (LC) diseases such as LC light-chain amyloidosis die with organ failure and need new therapies. We sought a model to test anti-LC siRNA delivery to human plasma cells, requiring circulating LC, in vivo indicators of tumor presence, and capacity for multiple injections of delivery vehicle. The JJN-3 human myeloma reporter cell line expressing firefly luciferase (FFL) implanted intraperitoneally (IP) in the NOD scid γ (NSG) mouse has a 90% prompt tumor-take, rapid LC production, and in vivo indicators of tumor measurable on day 5 post-implant (κ LC, bioluminescent signal, and soluble B-cell maturation antigen [sBCMA]) with median day 5 serum levels of κ LC of 1482 ng/mL (range, 255-4831) and robust correlations with all in vivo indicators. In preliminary attempts to deliver siRNA against κ LC constant region mRNA, we identified the 306-O18B3 lipidoid nanoparticle (LNP) as promising, safe and efficient in vitro. In vivo in the JJN-3 NSG IP model, after daily IP 306-O18B3:siRNA injections on days 5-10, a reduction in κ LC was observed on day 8 between control and test groups that continued through day 12 at sacrifice. This model is potentially useful as a platform for refining anti-LC therapies.


Asunto(s)
Técnicas de Transferencia de Gen , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/terapia , Cadenas kappa de Inmunoglobulina/genética , Tratamiento con ARN de Interferencia/métodos , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Linfocitos B/metabolismo , Línea Celular Tumoral , Células Cultivadas , Femenino , Humanos , Cadenas kappa de Inmunoglobulina/metabolismo , Luciferasas de Luciérnaga/genética , Luciferasas de Luciérnaga/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Nanopartículas/efectos adversos , Nanopartículas/química
18.
Biomacromolecules ; 20(9): 3333-3339, 2019 09 09.
Artículo en Inglés | MEDLINE | ID: mdl-31342740

RESUMEN

Genome-editing technologies hold tremendous potential for treating genetic diseases. However, the efficient and safe delivery of genome-editing elements to the location of interest, and the achievement of specific targeted gene correction without off-target side effect remains a big challenge. In this Perspective, we highlight recent developments and discuss the challenges of nonviral nanoparticles for the delivery of genome-editing tools. Finally, we will propose promising strategies to improve the delivery efficacy and advance the clinical translation of gene-editing technology.


Asunto(s)
Sistemas CRISPR-Cas/genética , Técnicas de Transferencia de Gen , Nanopartículas/química , Ácidos Nucleicos/genética , Edición Génica/métodos , Terapia Genética , Humanos , Nanopartículas/uso terapéutico , Ácidos Nucleicos/uso terapéutico
19.
Proc Natl Acad Sci U S A ; 113(11): 2868-73, 2016 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-26929348

RESUMEN

A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane, including escape from endosomes. Here we report that combining bioreducible lipid nanoparticles with negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives the electrostatic assembly of nanoparticles that mediate potent protein delivery and genome editing. These bioreducible lipids efficiently deliver protein cargo into cells, facilitate the escape of protein from endosomes in response to the reductive intracellular environment, and direct protein to its intracellular target sites. The delivery of supercharged Cre protein and Cas9:sgRNA complexed with bioreducible lipids into cultured human cells enables gene recombination and genome editing with efficiencies greater than 70%. In addition, we demonstrate that these lipids are effective for functional protein delivery into mouse brain for gene recombination in vivo. Therefore, the integration of this bioreducible lipid platform with protein engineering has the potential to advance the therapeutic relevance of protein-based genome editing.


Asunto(s)
Técnicas de Inactivación de Genes , Genes Sintéticos , Ingeniería Genética/métodos , Lípidos/química , Nanopartículas , Animales , Proteínas Bacterianas/administración & dosificación , Proteínas Bacterianas/genética , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Ceramidas/química , Colesterol/química , Portadores de Fármacos , Endocitosis , Endonucleasas/administración & dosificación , Endonucleasas/genética , Endosomas/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Hipotálamo/metabolismo , Integrasas/administración & dosificación , Integrasas/genética , Lípidos/administración & dosificación , Lípidos/síntesis química , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Ratones , Estructura Molecular , Nanopartículas/administración & dosificación , Nanopartículas/química , Nanopartículas/metabolismo , Nanopartículas/toxicidad , Fosfatidiletanolaminas/química , ARN/genética , Proteínas Recombinantes/biosíntesis , Recombinación Genética , Electricidad Estática , Relación Estructura-Actividad , Tálamo/metabolismo
20.
Int J Cancer ; 143(7): 1797-1805, 2018 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-29696632

RESUMEN

Gut dysbiosis may play an etiological role in colorectal tumorigenesis. We previously observed that the abundance of Parabacteroides distasonis (Pd) in stool was inversely associated with intestinal tumor burden and IL-1ß concentrations in mice. Here, we assessed the anti-inflammatory capacity of Pd membrane fraction (PdMb) in colon cancer cell lines. In addition, we tested whether Pd could suppress colon tumorigenesis in mice. Six-week-old male A/J mice were fed a low-fat (LF) diet, high-fat (HF) diet or HF+ whole freeze-dried Pd (HF + Pd, 0.04% wt/wt) for 24 weeks. After 1 week on diet, mice received 4 weekly injections of azoxymethane. PdMb robustly suppressed the production of pro-inflammatory cytokines and lowered the abundance of MyD88 and pAkt (ser473) induced by E. coli lipopolysaccharide in colon cancer cell lines. Moreover, PdMb induced apoptosis in colon cancer cell lines and blocked TLR4 activation in a reporter line. Colon tumors were observed in 0% of LF (0 of 19), 25% of HF (5 of 20) and 0% of HF + Pd mice (0 of 20) (p = 0.005). The latter group also displayed a lower abundance of MyD88 and pAkt (ser473) in colonic mucosa than HF mice. Taken together, these data suggest that Pd has anti-inflammatory and anti-cancer properties that are likely mediated by the suppression of TLR4 and Akt signaling, as well as promotion of apoptosis. Further work is needed to confirm these findings in additional models and fully elaborate the mechanism of action.


Asunto(s)
Azoximetano/toxicidad , Bacteroidetes/fisiología , Neoplasias del Colon/prevención & control , Dieta Alta en Grasa/efectos adversos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Apoptosis , Carcinógenos/toxicidad , Proliferación Celular , Neoplasias del Colon/etiología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Humanos , Masculino , Ratones , Ratones Endogámicos A , Células Tumorales Cultivadas
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