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1.
Exp Eye Res ; 240: 109789, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38242423

RESUMEN

Age-related macular degeneration (AMD), a leading cause of vision loss, primarily arises from the degeneration of retinal pigment epithelium (RPE) and photoreceptors. Current therapeutic options for dry AMD are limited. Encouragingly, cultured RPE cells on parylene-based biomimetic Bruch's membrane demonstrate characteristics akin to the native RPE layer. In this study, we cultivated human embryonic stem cell-derived polarized RPE (hESC-PRPE) cells on parylene membranes at both small- and large-scale settings, collecting conditioned supernatant, denoted as PRPE-SF. We conducted a comprehensive analysis of the morphology of the cultured hESC-RPE cells and the secreted growth factors in PRPE-SF. To evaluate the in vivo efficacy of these products, the product was administered via intravitreal injections of PRPE-SF in immunodeficient Royal College of Surgeons (iRCS) rats, a model for retinal degeneration. Our study not only demonstrated the scalability of PRPE-SF production while maintaining RPE cell phenotype but also showed consistent protein concentrations between small- and large-scale batches. We consistently identified 10 key factors in PRPE-SF, including BMP-7, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, MANF, PEDF, PDGF-AA, TGFß1, and VEGF. Following intravitreal administration of PRPE-SF, we observed a significant increase in the thickness of the outer nuclear layer (ONL) and photoreceptor preservation in iRCS rats. Furthermore, correlation analysis revealed that IGFBP-3, IGFBP-4, MANF, PEDF, and TGFß1 displayed positive associations with in vivo bioactivity, while GDF-15 exhibited a negative correlation. Overall, this study highlights the feasibility of scaling up PRPE-SF production on parylene membranes without compromising its essential constituents. The outcomes of PRPE-SF administration in an animal model of retinal degeneration present substantial potential for photoreceptor preservation. Moreover, the identification of candidate surrogate potency markers, showing strong positive associations with in vivo bioactivity, lays a solid foundation for the development of a promising therapeutic intervention for retinal degenerative diseases.


Asunto(s)
Polímeros , Degeneración Retiniana , Epitelio Pigmentado de la Retina , Xilenos , Humanos , Animales , Ratas , Epitelio Pigmentado de la Retina/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina , Degeneración Retiniana/metabolismo
2.
Ophthalmology ; 131(6): 682-691, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38160882

RESUMEN

PURPOSE: To report long-term results from a phase 1/2a clinical trial assessment of a scaffold-based human embryonic stem cell-derived retinal pigmented epithelium (RPE) implant in patients with advanced geographic atrophy (GA). DESIGN: A single-arm, open-label phase 1/2a clinical trial approved by the United States Food and Drug Administration. PARTICIPANTS: Patients were 69-85 years of age at the time of enrollment and were legally blind in the treated eye (best-corrected visual acuity [BCVA], ≤ 20/200) as a result of GA involving the fovea. METHODS: The clinical trial enrolled 16 patients, 15 of whom underwent implantation successfully. The implant was administered to the worse-seeing eye with the use of a custom subretinal insertion device. The companion nonimplanted eye served as the control. The primary endpoint was at 1 year; thereafter, patients were followed up at least yearly. MAIN OUTCOME MEASURES: Safety was the primary endpoint of the study. The occurrence and frequency of adverse events (AEs) were determined by scheduled eye examinations, including measurement of BCVA and intraocular pressure and multimodal imaging. Serum antibody titers were collected to monitor systemic humoral immune responses to the implanted cells. RESULTS: At a median follow-up of 3 years, fundus photography revealed no migration of the implant. No unanticipated, severe, implant-related AEs occurred, and the most common anticipated severe AE (severe retinal hemorrhage) was eliminated in the second cohort (9 patients) through improved intraoperative hemostasis. Nonsevere, transient retinal hemorrhages were noted either during or after surgery in all patients as anticipated for a subretinal surgical procedure. Throughout the median 3-year follow-up, results show that implanted eyes were more likely to improve by > 5 letters of BCVA and were less likely to worsen by > 5 letters compared with nonimplanted eyes. CONCLUSIONS: This report details the long-term follow-up of patients with GA to receive a scaffold-based stem cell-derived bioengineered RPE implant. Results show that the implant, at a median 3-year follow-up, is safe and well tolerated in patients with advanced dry age-related macular degeneration. The safety profile, along with the early indication of efficacy, warrants further clinical evaluation of this novel approach for the treatment of GA. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.


Asunto(s)
Atrofia Geográfica , Epitelio Pigmentado de la Retina , Agudeza Visual , Humanos , Atrofia Geográfica/cirugía , Atrofia Geográfica/fisiopatología , Epitelio Pigmentado de la Retina/trasplante , Epitelio Pigmentado de la Retina/patología , Anciano , Agudeza Visual/fisiología , Femenino , Anciano de 80 o más Años , Masculino , Estudios de Seguimiento , Tomografía de Coherencia Óptica , Células Madre Embrionarias Humanas/trasplante , Células Madre Embrionarias Humanas/citología , Trasplante de Células Madre , Resultado del Tratamiento
3.
Cells ; 12(13)2023 06 22.
Artículo en Inglés | MEDLINE | ID: mdl-37443724

RESUMEN

Retinal degenerative diseases, including age-related macular degeneration (AMD) and retinitis pigmentosa, lack effective therapies. Conventional monotherapeutic approaches fail to target the multiple affected pathways in retinal degeneration. However, the retinal pigment epithelium (RPE) secretes several neurotrophic factors addressing diverse cellular pathways, potentially preserving photoreceptors. This study explored human embryonic stem cell-derived, polarized RPE soluble factors (PRPE-SF) as a combination treatment for retinal degeneration. PRPE-SF promoted retinal progenitor cell survival, reduced oxidative stress in ARPE-19 cells, and demonstrated critical antioxidant and anti-inflammatory effects for preventing retinal degeneration in the Royal College of Surgeons (RCS) rat model. Importantly, PRPE-SF treatment preserved retinal structure and scotopic b-wave amplitudes, suggesting therapeutic potential for delaying retinal degeneration. PRPE-SF is uniquely produced using biomimetic membranes for RPE polarization and maturation, promoting a protective RPE secretome phenotype. Additionally, PRPE-SF is produced without animal serum to avoid immunogenicity in future clinical development. Lastly, PRPE-SF is a combination of neurotrophic factors, potentially ameliorating multiple dysfunctions in retinal degenerations. In conclusion, PRPE-SF offers a promising therapeutic candidate for retinal degenerative diseases, advancing the development of effective therapeutic strategies for these debilitating conditions.


Asunto(s)
Degeneración Retiniana , Epitelio Pigmentado de la Retina , Ratas , Humanos , Animales , Epitelio Pigmentado de la Retina/metabolismo , Degeneración Retiniana/metabolismo , Secretoma , Retina/metabolismo , Células Fotorreceptoras/metabolismo
4.
J Vis Exp ; (190)2022 12 02.
Artículo en Inglés | MEDLINE | ID: mdl-36533846

RESUMEN

Retinal degeneration, such as age-related macular degeneration (AMD), is a leading cause of blindness worldwide. A myriad of approaches have been undertaken to develop regenerative medicine-based therapies for AMD, including stem cell-based therapies. Rodents as animal models for retinal degeneration are a foundation for translational research, due to the broad spectrum of strains that develop retinal degeneration diseases at different stages. However, mimicking human therapeutic delivery of subretinal implants in rodents is challenging, due to anatomical differences such as lens size and vitreous volume. This surgical protocol aims to provide a guided method for transplanting implants into the subretinal space in rats. A user-friendly comprehensive description of the critical steps has been included. This protocol has been developed as a cost-efficient surgical procedure for reproducibility across different preclinical studies in rats. Proper miniaturization of a human-sized implant is required prior to conducting the surgical experiment, which includes adjustments to the dimensions of the implant. An external approach is used instead of an intravitreal procedure to deliver the implant to the subretinal space. Using a small sharp needle, a scleral incision is performed in the temporal superior quadrant, followed by paracentesis to reduce intraocular pressure, thereby minimizing resistance during the surgical implantation. Next, a balanced salt solution (BSS) injection through the incision is carried out to achieve focal retinal detachment (RD). Lastly, insertion and visualization of the implant into the subretinal space are conducted. Post-operative assessment of the subretinal placement of the implant includes imaging by spectral domain optical coherence tomography (SD-OCT). Imaging follow-ups ascertain the subretinal stability of the implant, before the eyes are harvested and fixated for histological analysis.


Asunto(s)
Degeneración Macular , Degeneración Retiniana , Humanos , Ratas , Animales , Degeneración Retiniana/cirugía , Degeneración Retiniana/patología , Reproducibilidad de los Resultados , Modelos Animales de Enfermedad , Degeneración Macular/terapia , Tomografía de Coherencia Óptica/métodos
5.
Stem Cell Reports ; 17(3): 448-458, 2022 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-35120620

RESUMEN

Cell-based therapies face challenges, including poor cell survival, immune rejection, and integration into pathologic tissue. We conducted an open-label phase 1/2a clinical trial to assess the safety and preliminary efficacy of a subretinal implant consisting of a polarized monolayer of allogeneic human embryonic stem cell-derived retinal pigmented epithelium (RPE) cells in subjects with geographic atrophy (GA) secondary to dry age-related macular degeneration. Postmortem histology from one subject with very advanced disease shows the presence of donor RPE cells 2 years after implantation by immunoreactivity for RPE65 and donor-specific human leukocyte antigen (HLA) class I molecules. Markers of RPE cell polarity and phagocytosis suggest donor RPE function. Further histologic examination demonstrated CD34+ structures beneath the implant and CD4+, CD68+, and FoxP3+ cells in the tissue. Despite significant donor-host HLA mismatch, no clinical signs of retinitis, vitreitis, vasculitis, choroiditis, or serologic immune response were detected in the deceased subject or any other subject in the study. Subretinally implanted, HLA-mismatched donor RPE cells survive, express functional markers, and do not elicit clinically detectable intraocular inflammation or serologic immune responses even without long-term immunosuppression.


Asunto(s)
Atrofia Geográfica , Degeneración Macular , Prótesis e Implantes , Atrofia Geográfica/terapia , Células Madre Embrionarias Humanas/patología , Humanos , Degeneración Macular/patología , Degeneración Macular/terapia , Prótesis e Implantes/efectos adversos , Epitelio Pigmentado de la Retina/patología
6.
Transl Vis Sci Technol ; 10(10): 13, 2021 08 12.
Artículo en Inglés | MEDLINE | ID: mdl-34613357

RESUMEN

Purpose: To report 1-year follow-up of a phase 1/2a clinical trial testing a composite subretinal implant having polarized human embryonic stem cell (hESC)-derived retinal pigment epithelium (RPE) cells on an ultrathin parylene substrate in subjects with advanced non-neovascular age-related macular degeneration (NNAMD). Methods: The phase 1/2a clinical trial included 16 subjects in two cohorts. The main endpoint was safety assessed at 365 days using ophthalmic and systemic exams. Pseudophakic subjects with geographic atrophy (GA) and severe vision loss were eligible. Low-dose tacrolimus immunosuppression was utilized for 68 days in the peri-implantation period. The implant was delivered to the worst seeing eye with a custom subretinal insertion device in an outpatient setting. A data safety monitoring committee reviewed all results. Results: The treated eyes of all subjects were legally blind with a baseline best-corrected visual acuity (BCVA) of ≤ 20/200. There were no unexpected serious adverse events. Four subjects in cohort 1 had serious ocular adverse events, including retinal hemorrhage, edema, focal retinal detachment, or RPE detachment, which was mitigated in cohort 2 using improved hemostasis during surgery. Although this study was not powered to assess efficacy, treated eyes from four subjects showed an increased BCVA of >5 letters (6-13 letters). A larger proportion of treated eyes experienced a >5-letter gain when compared with the untreated eye (27% vs. 7%; P = not significant) and a larger proportion of nonimplanted eyes demonstrated a >5-letter loss (47% vs. 33%; P = not significant). Conclusions: Outpatient delivery of the implant can be performed routinely. At 1 year, the implant is safe and well tolerated in subjects with advanced dry AMD. Translational Relevance: This work describes the first clinical trial, to our knowledge, of a novel implant for advanced dry AMD.


Asunto(s)
Atrofia Geográfica , Trasplante de Células Madre Hematopoyéticas , Degeneración Macular , Estudios de Seguimiento , Atrofia Geográfica/terapia , Humanos , Degeneración Macular/terapia , Agudeza Visual
7.
Sci Rep ; 11(1): 6286, 2021 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-33737600

RESUMEN

Age-related macular degeneration (AMD) is the primary cause of blindness in adults over 60 years of age, and clinical trials are currently assessing the therapeutic potential of retinal pigmented epithelial (RPE) cell monolayers on implantable scaffolds to treat this disease. However, challenges related to the culture, long-term storage, and long-distance transport of such implants currently limit the widespread use of adherent RPE cells as therapeutics. Here we report a xeno-free protocol to cryopreserve a confluent monolayer of clinical-grade, human embryonic stem cell-derived RPE cells on a parylene scaffold (REPS) that yields viable, polarized, and functional RPE cells post-thaw. Thawed cells exhibit ≥ 95% viability, have morphology, pigmentation, and gene expression characteristic of mature RPE cells, and secrete the neuroprotective protein, pigment epithelium-derived factor (PEDF). Stability under liquid nitrogen (LN2) storage has been confirmed through one year. REPS were administered immediately post-thaw into the subretinal space of a mammalian model, the Royal College of Surgeons (RCS)/nude rat. Implanted REPS were assessed at 30, 60, and 90 days post-implantation, and thawed cells demonstrate survival as an intact monolayer on the parylene scaffold. Furthermore, immunoreactivity for the maturation marker, RPE65, significantly increased over the post-implantation period in vivo, and cells demonstrated functional attributes similar to non-cryopreserved controls. The capacity to cryopreserve adherent cellular therapeutics permits extended storage and stable transport to surgical sites, enabling broad distribution for the treatment of prevalent diseases such as AMD.


Asunto(s)
Criopreservación/métodos , Células Epiteliales/trasplante , Degeneración Macular/terapia , Epitelio Pigmentado de la Retina/trasplante , Manejo de Especímenes/métodos , Trasplante de Células Madre/métodos , Animales , Diferenciación Celular , Línea Celular , Supervivencia Celular , Modelos Animales de Enfermedad , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteínas del Ojo/metabolismo , Células Madre Embrionarias Humanas/citología , Humanos , Factores de Crecimiento Nervioso/metabolismo , Polímeros , Ratas , Ratas Desnudas , Medicina Regenerativa/métodos , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Serpinas/metabolismo , Andamios del Tejido , Resultado del Tratamiento , Xilenos
8.
Sci Rep ; 9(1): 11230, 2019 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-31375713

RESUMEN

Patients diagnosed with Retinitis Pigmentosa (RP) show, in the advanced stage of the disease, severely restricted peripheral vision causing poor mobility and decline in quality of life. This vision loss causes difficulty identifying obstacles and their relative distances. Thus, RP patients use mobility aids such as canes to navigate, especially in dark environments. A number of high-tech visual aids using virtual reality (VR) and sensory substitution have been developed to support or supplant traditional visual aids. These have not achieved widespread use because they are difficult to use or block off residual vision. This paper presents a unique depth to high-contrast pseudocolor mapping overlay developed and tested on a Microsoft Hololens 1 as a low vision aid for RP patients. A single-masked and randomized trial of the AR pseudocolor low vision aid to evaluate real world mobility and near obstacle avoidance was conducted consisting of 10 RP subjects. An FDA-validated functional obstacle course and a custom-made grasping setup were used. The use of the AR visual aid reduced collisions by 50% in mobility testing (p = 0.02), and by 70% in grasp testing (p = 0.03). This paper introduces a new technique, the pseudocolor wireframe, and reports the first significant statistics showing improvements for the population of RP patients with mobility and grasp.


Asunto(s)
Recursos Audiovisuales , Realidad Aumentada , Baja Visión/rehabilitación , Accidentes por Caídas/prevención & control , Adulto , Femenino , Humanos , Masculino , Calidad de Vida , Realidad Virtual
9.
Sci Transl Med ; 10(435)2018 04 04.
Artículo en Inglés | MEDLINE | ID: mdl-29618560

RESUMEN

Retinal pigment epithelium (RPE) dysfunction and loss are a hallmark of non-neovascular age-related macular degeneration (NNAMD). Without the RPE, a majority of overlying photoreceptors ultimately degenerate, leading to severe, progressive vision loss. Clinical and histological studies suggest that RPE replacement strategies may delay disease progression or restore vision. A prospective, interventional, U.S. Food and Drug Administration-cleared, phase 1/2a study is being conducted to assess the safety and efficacy of a composite subretinal implant in subjects with advanced NNAMD. The composite implant, termed the California Project to Cure Blindness-Retinal Pigment Epithelium 1 (CPCB-RPE1), consists of a polarized monolayer of human embryonic stem cell-derived RPE (hESC-RPE) on an ultrathin, synthetic parylene substrate designed to mimic Bruch's membrane. We report an interim analysis of the phase 1 cohort consisting of five subjects. Four of five subjects enrolled in the study successfully received the composite implant. In all implanted subjects, optical coherence tomography imaging showed changes consistent with hESC-RPE and host photoreceptor integration. None of the implanted eyes showed progression of vision loss, one eye improved by 17 letters and two eyes demonstrated improved fixation. The concurrent structural and functional findings suggest that CPCB-RPE1 may improve visual function, at least in the short term, in some patients with severe vision loss from advanced NNAMD.


Asunto(s)
Degeneración Macular/terapia , Células Cultivadas , Femenino , Atrofia Geográfica/terapia , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/fisiología , Humanos , Masculino , Estudios Prospectivos , Epitelio Pigmentado de la Retina/citología , Trasplante de Células Madre , Tomografía de Coherencia Óptica
10.
Acta Biomater ; 9(8): 7796-805, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23619290

RESUMEN

We have previously shown that recombinant human collagen can be crosslinked with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) to fabricate transparent hydrogels possessing the shape and dimensions of the human cornea. These corneal implants have been tested in a Phase I human clinical study. Although these hydrogels successfully promoted corneal tissue and nerve regeneration, the gelling kinetics were difficult to control during the manufacture of the implants. An alternative carbodiimide capable of producing hydrogels of similar characteristics as EDC in terms of strength and biocompatibility, but with a longer gelation time would be a desirable alternative. Here, we compared the crosslinking kinetics and properties of hydrogels crosslinked with a sterically bulky carbodiimide, N-Cyclohexyl-N'-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMC), with that of EDC. CMC crosslinking was possible at ambient temperature whereas the EDC reaction was too rapid to control and had to be carried out at low temperatures. The highest tensile strength obtained using optimized formulations were equivalent, although CMC crosslinked hydrogels were found to be stiffer. The collagenase resistance of CMC crosslinked hydrogels was superior to that of EDC crosslinked hydrogels while biocompatibility was similar. We are also able to substitute porcine collagen with recombinant human collagen and show that the in vivo performance of both resulting hydrogels as full-thickness corneal implants is comparable in a mouse model of an orthotopic corneal graft. In conclusion, CMC is a viable alternative to EDC as a crosslinker for collagen-based biomaterials for use as corneal implants, and potentially for use in other tissue engineering applications.


Asunto(s)
Carbodiimidas/química , Colágeno Tipo I/química , Córnea/cirugía , Trasplante de Córnea/instrumentación , Reactivos de Enlaces Cruzados/química , Hidrogeles/química , Prótesis e Implantes , Animales , Córnea/citología , Módulo de Elasticidad , Diseño de Equipo , Análisis de Falla de Equipo , Dureza , Ensayo de Materiales , Ratones , Ratones Endogámicos BALB C , Porcinos , Resistencia a la Tracción , Resultado del Tratamiento
11.
J Funct Biomater ; 4(3): 162-77, 2013 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-24956085

RESUMEN

A bi-functional epoxy-based cross-linker, 1,4-Butanediol diglycidyl ether (BDDGE), was investigated in the fabrication of collagen based corneal substitutes. Two synthetic strategies were explored in the preparation of the cross-linked collagen scaffolds. The lysine residues of Type 1 porcine collagen were directly cross-linked using l,4-Butanediol diglycidyl ether (BDDGE) under basic conditions at pH 11. Alternatively, under conventional methodology, using both BDDGE and 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) as cross-linkers, hydrogels were fabricated under acidic conditions. In this latter strategy, Cu(BF4)2·XH2O was used to catalyze the formation of secondary amine bonds. To date, we have demonstrated that both methods of chemical cross-linking improved the elasticity and tensile strength of the collagen implants. Differential scanning calorimetry and biocompatibility studies indicate comparable, and in some cases, enhanced properties compared to that of the EDC/NHS controls. In vitro studies showed that human corneal epithelial cells and neuronal progenitor cell lines proliferated on these hydrogels. In addition, improvement of cell proliferation on the surfaces of the materials was observed when neurite promoting laminin epitope, IKVAV, and adhesion peptide, YIGSR, were incorporated. However, the elasticity decreased with peptide incorporation and will require further optimization. Nevertheless, we have shown that epoxy cross-linkers should be further explored in the fabrication of collagen-based hydrogels, as alternatives to or in conjunction with carbodiimide cross-linkers.

12.
Biomaterials ; 30(29): 5403-8, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19576630

RESUMEN

Saturated neoglycopolymers, prepared via tandem ROMP-hydrogenation (ROMP=ring-opening metathesis polymerization) of carbohydrate-functionalized norbornenes, are investigated as novel collagen crosslinking agents in corneal tissue engineering. The neoglycopolymers were incorporated into recombinant human collagen type III (RHC III) as collagen crosslinking agents and glycosaminoglycan (GAG) mimics. The purely synthetic nature of these composites is designed to reduce susceptibility to immunological and allergic reactions, and to circumvent the transmission of animal infectious diseases. The collagen-neoglycopolymer biomaterials exhibit higher stability to collagenase-induced biodegradation than the control materials, composites of RHC III crosslinked using EDC/NHS (EDC=1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide; NHS=N-hydroxysuccinimide). Even at this proof of concept stage, the thermal stability, enzymatic resistance, and permeability of the neoglycopolymer hydrogels are comparable or superior to those of these fully optimized control materials, which have successfully been tested clinically. Tensile strength is adequate for transplantation, but lower than that of the optimized control materials.


Asunto(s)
Materiales Biocompatibles/química , Materiales Biomiméticos/química , Colágeno/química , Córnea/crecimiento & desarrollo , Polímeros/química , Líquidos Corporales/química , Reactivos de Enlaces Cruzados/química , Módulo de Elasticidad , Humanos , Ensayo de Materiales , Proteínas Recombinantes/química
13.
J Am Chem Soc ; 131(24): 8496-502, 2009 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-19485360

RESUMEN

We describe a synthetic strategy that permits both the growth and deprotection of RNA chains that remain attached to a solid polymer support or chip surface. The key synthons for RNA synthesis are novel 5'-O-DMTr 2'-acetal levulinyl ester (2'-O-ALE) ribonucleoside 3'-phosphoramidite derivatives. In the presence of 4,5-dicyanoimidazole (DCI) as the activator, these monomers coupled to Q-CPG solid support with excellent coupling efficiency (approximately 98.7%). The method was extended to the light directed synthesis of poly rU and poly rA on a microarray through the use of a 5'-O-(2-(2-nitrophenyl)propoxycarbonyl)-2'-O-ALE-3'-phosphoramidite derivative. A two-stage deprotection strategy was employed to fully deblock the RNA directly on the Q-CPG or microarray support without releasing it from the support's surface: phosphate group deblocking with NEt(3) in acetonitrile (ACN) (2:3 v/v; 1 h, r.t.) followed by removal of the 2'-O-ALE groups under mild hydrazinolysis conditions (0.5-4 h, r.t.). This last treatment also removed the levulinyl (Lv) group on adenine (N(6)) and cytosine (N(4)) and the dimethylformamidine (dmf) group on guanine (N(2)). The chemistry and methods described here pave the way to the fabrication of microarrays of immobilized RNA probes for analyzing molecular interactions of biological interest.


Asunto(s)
Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Oligorribonucleótidos/síntesis química , Ribonucleósidos/química , Acetales/química , Ésteres/química , Ácidos Levulínicos/química , Compuestos Organofosforados/química , ARN sin Sentido/química
14.
J Org Chem ; 72(25): 9491-500, 2007 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-17979285

RESUMEN

Current studies of lariat RNA structure and function are hindered by the lack of access to synthetic lariats. A novel approach to the synthesis of both DNA and RNA lariats is presented here. Noteworthy features of the methodology are the regiospecific formation of the 2'-5'-phosphodiester linkage, the unusual parallel stranded DNA/RNA hybrid (or parallel RNA/RNA duplex) that forms between an RNA template and a folded 22-nt DNA (or RNA) substrate, and the efficiency of the chemical ligation step at an adenosine branchpoint (50-80%). The DNA and RNA lariats were purified by polyacrylamide gel electrophoresis, and their structure and nucleotide composition were confirmed by MALDI-TOF mass spectrometry. Thermal denaturation as well as enzymatic and chemical hydrolysis fully supported the proposed lariat structures. Characterization of control parallel duplexes was conducted by gel shift assays and enzymatic degradation with RNase H. The successful synthesis of the lariat molecules described here will allow structural and biochemical studies aimed at better understanding the splicing and debranching mechanisms in which these unusual nucleic acids are involved.


Asunto(s)
ADN Circular/síntesis química , ARN/síntesis química , ADN Circular/química , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , ARN/química , ARN Circular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Temperatura
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