ABSTRACT
OBJECTIVE: Current gene therapy of inherited retinal diseases is achieved mainly by subretinal injection, which is invasive with severe adverse effects. Intravitreal injection is a minimally invasive alternative for gene therapy of inherited retinal diseases. This work explores the efficacy of intravitreal delivery of PEGylated ECO (a multifunctional pH-sensitive amphiphilic amino lipid) plasmid DNA (pGRK1-ABCA4-S/MAR) nanoparticles (PEG-ELNP) for gene therapy of Stargardt disease. METHODS: Pigmented Abca4-/- knockout mice received 1 µL of PEG-ELNP solution (200 ng/uL, pDNA concentration) by intravitreal injections at an interval of 1.5 months. The expression of ABCA4 in the retina was determined by RT-PCR and immunohistochemistry at 6 months after the second injection. A2E levels in the treated eyes and untreated controls were determined by HPLC. The safety of treatment was monitored by scanning laser ophthalmoscopy and electroretinogram (ERG). RESULTS: PEG-ELNP resulted in significant ABCA4 expression at both mRNA level and protein level at]6 months after 2 intravitreal injections, and a 40% A2E accumulation reduction compared with non-treated controls. The PEG-ELNP also demonstrated excellent safety as shown by scanning laser ophthalmoscopy, and the eye function evaluation from electroretinogram. CONCLUSIONS: Intravitreal delivery of the PEG-ELNP of pGRK1-ABCA4-S/MAR is a promising approach for gene therapy of Stargardt Disease, which can also be a delivery platform for gene therapy of other inherited retinal diseases.
Subject(s)
Nanoparticles , Retina , Mice , Animals , Stargardt Disease/genetics , Stargardt Disease/metabolism , Stargardt Disease/therapy , Retina/metabolism , Genetic Therapy/methods , Plasmids/genetics , DNA/metabolism , Mice, Knockout , Polyethylene Glycols/metabolism , Intravitreal Injections , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolismABSTRACT
Stargardt disease (STGD) is the most common form of inherited retinal genetic disorders and is often caused by mutations in ABCA4. Gene therapy has the promise to effectively treat monogenic retinal disorders. However, clinically approved adeno-associated virus (AAV) vectors do not have a loading capacity for large genes, such as ABCA4. Self-assembly nanoparticles composed of (1-aminoethyl)iminobis[N-(oleoylcysteinyl-1-amino-ethyl)propionamide (ECO; a multifunctional pH-sensitive/ionizable amino lipid) and plasmid DNA produce gene transfection comparable with or better than the AAV2 capsid. Stable PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles produce specific and prolonged expression of ABCA4 in the photoreceptors of Abca4 -/- mice and significantly inhibit accumulation of toxic A2E in the eye. Multiple subretinal injections enhance gene expression and therapeutic efficacy with an approximately 69% reduction in A2E accumulation in Abca4 -/- mice after 3 doses. Very mild inflammation was observed after multiple injections of the nanoparticles. PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles are a promising non-viral mediated gene therapy modality for STGD type 1 (STGD1).
ABSTRACT
Safe and effective molecular therapeutics for prophylactic treatment of retinal degenerative diseases are greatly needed. Disruptions in the clearance of all-trans-retinal (atRAL) by the visual (retinoid) cycle of the retina can lead to the accumulation of atRAL and its condensation products known to initiate progressive retinal dystrophy. Retinylamine (Ret-NH2) and its analogues are known to be effective in lowering the concentration of atRAL within the eye and thus preventing retinal degeneration in mouse models of human retinopathies. Here, we chemically modified Ret-NH2 with amino acids and peptides to improve the stability and ocular bioavailability of the resulting derivatives and to minimize their side effects. Fourteen Ret-NH2 derivatives were synthesized and tested in vitro and in vivo. These derivatives exhibited structure-dependent therapeutic efficacy in preventing light-induced retinal degeneration in Abca4-/-Rdh8-/- double-knockout mice, with the compounds containing glycine and/or L-valine generally exhibiting greater protective effects than Ret-NH2 or other tested amino acid derivatives of Ret-NH2. Ret-NH2-L-valylglycine amide (RVG) exhibited good stability in storage; and effective uptake and prolonged retention in mouse eyes. RVG readily formed a Schiff base with atRAL and did not inhibit RPE65 enzymatic activity. Administered by oral gavage, this retinoid also provided effective protection against light-induced retinal degeneration in Abca4-/-Rdh8-/- mice. Notably, the treatment with RVG had minimal effects on the regeneration of 11-cis-retinal and recovery of retinal function. RVG holds promise as a lead therapy for effective and safe treatment of human retinal degenerative diseases.
Subject(s)
Diterpenes/pharmacology , Peptides/pharmacology , Retinal Degeneration/prevention & control , Vision, Ocular/drug effects , ATP-Binding Cassette Transporters/genetics , Alcohol Oxidoreductases/genetics , Animals , Diterpenes/chemistry , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Retinal Degeneration/physiopathologyABSTRACT
It is still a challenge to develop gene replacement therapy for retinal disorders caused by mutations in large genes, such as Stargardt disease (STGD). STGD is caused by mutations in ABCA4 gene. Previously, we have developed an effective non-viral gene therapy using self-assembled nanoparticles of a multifunctional pH-sensitive amino lipid ECO and a therapeutic ABCA4 plasmid containing rhodopsin promoter (pRHO-ABCA4). In this study, we modified the ABCA4 plasmid with simian virus 40 enhancer (SV40, pRHO-ABCA4-SV40) for enhanced gene expression. We also prepared and assessed the formulations of ECO/pDNA nanoparticles using sucrose or sorbitol as a stablilizer to develop consistent and stable formulations. Results demonstrated that ECO formed stable nanoparticles with pRHO-ABCA4-SV40 in the presence of sucrose, but not with sorbitol. The transfection efficiency in vitro increased significantly after introduction of SV40 enhancer for plasmid pCMV-ABCA4-SV40 with a CMV promoter. Sucrose didn't affect the transfection efficiency, while sorbitol resulted in a fluctuation of the in vitro transfection efficiency. Subretinal gene therapy in Abca4-/- mice using ECO/pRHO-ABCA4 and ECO/pRHO-ABCA4-SV40 nanoparticles induced 36% and 29% reduction in A2E accumulation respectively. Therefore, the ECO/pABCA4 based nanoparticles are promising for non-viral gene therapy for Stargardt disease and can be expended for applications in a variety of visual dystrophies with mutated large genes.
Subject(s)
Nanoparticles , Simian virus 40 , ATP-Binding Cassette Transporters/genetics , Animals , Genetic Therapy , Mice , Mutation , Stargardt DiseaseABSTRACT
Stargardt disease (STGD) is an autosomal recessive retinal disorder caused by a monogenic ABCA4 mutation. Currently, there is no effective therapy to cure Stargardt disease. The replacement of mutated ABCA4 with a functional gene remains an attractive strategy. In this study, we have developed a non-viral gene therapy using nanoparticles self-assembled by a multifunctional pH-sensitive amino lipid ECO and a therapeutic ABCA4 plasmid. The nanoparticles mediated efficient intracellular gene transduction in wild-type (WT) and Abca4-/- mice. Specific ABCA4 expression in the outer segment of photoreceptors was achieved by incorporating a rhodopsin promoter into the plasmids. The ECO/pRHO-ABCA4 nanoparticles induced substantial and specific ABCA4 expression for at least 8 months, 35% reduction in A2E accumulation on average, and a delayed Stargardt disease progression for at least 6 months in Abca4-/- mice. ECO/plasmid nanoparticles constitute a promising non-viral gene therapy platform for Stargardt disease and other visual dystrophies.
Subject(s)
ATP-Binding Cassette Transporters/administration & dosage , ATP-Binding Cassette Transporters/metabolism , Drug Delivery Systems/methods , Genetic Therapy/methods , Lipopeptides/administration & dosage , Nanoparticles/chemistry , Rhodopsin/administration & dosage , Stargardt Disease/therapy , ATP-Binding Cassette Transporters/genetics , Animals , Cell Line , Disease Models, Animal , Humans , Lipopeptides/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Photoreceptor Cells/metabolism , Plasmids/genetics , Plasmids/therapeutic use , Retinal Pigment Epithelium/cytology , Retinal Pigment Epithelium/metabolism , Rhodopsin/genetics , Stargardt Disease/genetics , TransfectionABSTRACT
Retinal degeneration impairs the vision of millions in all age groups worldwide. Increasing evidence suggests that the etiology of many retinal degenerative diseases is associated with impairment in biochemical reactions involved in the visual cycle, a metabolic pathway responsible for regeneration of the visual chromophore (11-cis-retinal). Inefficient clearance of toxic retinoid metabolites, especially all-trans-retinal, is considered responsible for photoreceptor cytotoxicity. Primary amines, including retinylamine, are effective in lowing the concentration of all-trans-retinal within the retina and thus prevent retina degeneration in mouse models of human retinopathies. Here we achieved prolonged prevention of retinal degeneration by controlled delivery of retinylamine to the eye from polylactic acid nanoparticles in Abca4(-/-)Rdh8(-/-) (DKO) mice, an animal model of Stargardt disease/age-related macular degeneration. Subcutaneous administration of the nanoparticles containing retinylamine provided a constant supply of the drug to the eye for about a week and resulted in effective prolonged prevention of light-induced retinal degeneration in DKO mice. Retinylamine nanoparticles hold promise for prolonged prophylactic treatment of human retinal degenerative diseases, including Stargardt disease and age-related macular degeneration.
Subject(s)
Diterpenes/therapeutic use , Nanoparticles/therapeutic use , Retinal Degeneration/prevention & control , Animals , Diterpenes/pharmacokinetics , Diterpenes/pharmacology , Electroretinography , Humans , Immunohistochemistry , Lactic Acid/chemistry , Liver/drug effects , Liver/metabolism , Mice, Knockout , Nanoparticles/ultrastructure , Particle Size , Polyesters , Polymers/chemistry , Retinal Degeneration/drug therapy , Retinal Degeneration/physiopathology , Tomography, Optical CoherenceABSTRACT
A polyethylene glycol (PEG) retinylamine (Ret-NH2) conjugate PEG-GFL-NH-Ret with a glycine-phenylalanine-leucine (GFL) spacer was synthesized for controlled oral delivery of Ret-NH2 to treat retinal degenerative diseases, including Stargardt disease (STGD) and age-related macular degeneration (AMD). The peptide spacer was introduced for sustained release of the drug by digestive enzymes in the gastrointestinal tract. The pharmacokinetics experiments showed that the PEG conjugate could control the sustained drug release after oral administration and had much lower nonspecific liver drug accumulation than the free drug in wild-type female C57BL mice. In the mean time, the conjugate maintained the same concentration of Ret-NH2 in the eye as the free drug. Also, PEG-GFL-NH-Ret at a Ret-NH2 equivalent dose of 25 mg/kg produced complete protection of Abca4(-/-)Rdh8(-/-) mouse retinas against light-induced retinal degeneration for 3 days after oral administration, as revealed by OCT retina imaging, whereas free Ret-NH2 did not provide any protection under identical conditions. The polymer conjugate PEG-GFL-NH-Ret has great potential for controlled delivery of Ret-NH2 to the eye for effective protection against retinal degenerative diseases.
Subject(s)
Diterpenes/chemistry , Polyethylene Glycols/chemistry , Retinal Degeneration/drug therapy , Animals , Diterpenes/pharmacokinetics , Drug Delivery Systems , Female , Light/adverse effects , Macular Degeneration/congenital , Macular Degeneration/drug therapy , Macular Degeneration/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Polyethylene Glycols/pharmacokinetics , Retinal Degeneration/pathology , Stargardt DiseaseABSTRACT
PURPOSE: To design and develop a drug-delivery system containing a combination of poly(D,L-lactide-co-glycolide) (PLGA) microparticles and alginate hydrogel for sustained release of retinoids to treat retinal blinding diseases that result from an inadequate supply of retinol and generation of 11-cis-retinal. METHODS: To study drug release in vivo, either the drug-loaded microparticle-hydrogel combination was injected subcutaneously or drug-loaded microparticles were injected intravitreally into Lrat(-/-) mice. Orally administered 9-cis-retinoids were used for comparison and drug concentrations in plasma were determined by HPLC. Electroretinography (ERG) and both chemical and histologic analyses were used to evaluate drug effects on visual function and morphology. RESULTS: Lrat(-/-) mice demonstrated sustained drug release from the microparticle/hydrogel combination that lasted 4 weeks after subcutaneous injection. Drug concentrations in plasma of the control group treated with the same oral dose rose to higher levels for 6-7 hours but then dropped markedly by 24 hours. Significantly increased ERG responses and a markedly improved retinal pigmented epithelium (RPE)-rod outer segment (ROS) interface were observed after subcutaneous injection of the drug-loaded delivery combination. Intravitreal injection of just 2% of the systemic dose of drug-loaded microparticles provided comparable therapeutic efficacy. CONCLUSIONS: Sustained release of therapeutic levels of 9-cis-retinoids was achieved in Lrat(-/-) mice by subcutaneous injection in a microparticle/hydrogel drug-delivery system. Both subcutaneous and intravitreal injections of drug-loaded microparticles into Lrat(-/-) mice improved visual function and retinal structure.
Subject(s)
Blindness/drug therapy , Drug Delivery Systems/methods , Hydrogel, Polyethylene Glycol Dimethacrylate/pharmacology , Retinoids/pharmacology , Acyltransferases/genetics , Administration, Oral , Alginates/chemistry , Alginates/pharmacology , Animals , Blindness/genetics , Delayed-Action Preparations/pharmacology , Disease Models, Animal , Diterpenes , Drug Therapy, Combination , Electroretinography/drug effects , Female , Glucuronic Acid/chemistry , Glucuronic Acid/pharmacology , Hexuronic Acids/chemistry , Hexuronic Acids/pharmacology , Humans , Hydrogel, Polyethylene Glycol Dimethacrylate/chemistry , Injections, Subcutaneous , Intravitreal Injections , Lactic Acid/chemistry , Lactic Acid/pharmacology , Male , Mice , Mice, Mutant Strains , Microspheres , Polyglycolic Acid/chemistry , Polyglycolic Acid/pharmacology , Polylactic Acid-Polyglycolic Acid Copolymer , Retinaldehyde/metabolismABSTRACT
The antitumor activity of a colon-specific N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer - 9-aminocamptothecin (9-AC) conjugate (P-9-AC) was assessed in orthotopic and subcutaneous animal (HT29 xenograft) tumor models. P-9-AC treatment of mice bearing orthotopic colon tumors, with a dose of 3 mg/kg of 9-AC equivalent every other day for 6 weeks, resulted in regression of tumors in 9 of 10 mice. A lower dose of P-9-AC (1.25 mg/kg of 9-AC equivalent) every other day for 8 weeks inhibited subcutaneous tumor growth in all mice. No liver metastases were observed. Colon-specific release of 9-AC from polymer conjugates enhanced antitumor activity and minimized the systemic toxicity.
Subject(s)
Antineoplastic Agents , Camptothecin/analogs & derivatives , Colonic Neoplasms/drug therapy , Methacrylates , Polymers , Transplantation, Heterologous , Xenograft Model Antitumor Assays/methods , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Biocompatible Materials/chemistry , Biocompatible Materials/metabolism , Camptothecin/chemistry , Camptothecin/therapeutic use , Cell Line, Tumor , Drug Carriers/chemistry , Drug Carriers/metabolism , Drug Delivery Systems/methods , Female , Humans , Male , Materials Testing , Methacrylates/chemistry , Methacrylates/metabolism , Mice , Mice, Nude , Molecular Structure , Neoplasm Transplantation , Polymers/chemistry , Polymers/metabolism , Survival RateABSTRACT
PURPOSE: To quantitate and predict colon-specific 9-aminocamptothecin (9-AC) release from the N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-9-AC conjugate and its absorption behavior after oral administration in rats. METHODS: Drug distribution in the gastrointestinal (GI) tract and the plasma concentration-time profile of 9-AC released from the HPMA copolymer conjugate were predicted using the degradation, transit, and absorption rate constants in cecum. The fate of 9-AC in cecum and liver was measured by in-situ cecum absorption and liver perfusion. RESULTS: Following oral administration of the conjugate, 9-AC was released rapidly in cecum. Based on the pharmacokinetic model, up to 60% of the dose was in the cecum at ~6 h, and 7% of the dose still remained there at 24 h. The predicted plasma concentration curve for released 9-AC after an oral dose of 3 mg/kg of 9-AC equivalent increased gradually and reached a peak of 98 nM at 7 h, then started decreasing slowly to 16 nM at 24 h. The bioavailability value was estimated as 0.31 after the first-pass elimination. CONCLUSIONS: A pharmacokinetic model delineated the impact of GI transit, drug absorption rate, and first-pass metabolism on drug disposition following oral administration of HPMA copolymer-9-AC conjugate in rats.
Subject(s)
Antineoplastic Agents, Phytogenic/administration & dosage , Antineoplastic Agents, Phytogenic/pharmacokinetics , Camptothecin/analogs & derivatives , Colon/metabolism , Methacrylates/chemistry , Algorithms , Anesthetics, Local/pharmacokinetics , Animals , Antineoplastic Agents, Phytogenic/chemistry , Camptothecin/administration & dosage , Camptothecin/chemistry , Camptothecin/pharmacokinetics , Cecum/metabolism , Drug Delivery Systems , Gastrointestinal Transit , Indicators and Reagents , Injections, Intravenous , Intestinal Absorption , Lidocaine/pharmacokinetics , Liver/metabolism , Male , Models, Statistical , Protein Binding , Rats , Rats, WistarABSTRACT
A water soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-9-aminocamptothecin (9-AC) conjugate was designed for oral colon-specific drug delivery in the treatment of colon cancer. Comparative studies between the polymer conjugate and free drug have been performed to assess their biodistribution and pharmacokinetics in mice. After oral administration of equal doses of the polymer conjugate or free 9-AC, the drug concentrations in major organs at fixed time points were determined using an HPLC-fluorescence assay. Only 2+/-1% of 9-AC released from the polymer conjugate was detected in the small intestine (SI), and the mean peak concentration of free 9-AC was 45-fold higher than that from released drug. Colon-specific release of 9-AC produced high local concentrations. The mean peak concentration of released 9-AC in cecal contents, feces, cecal tissue, and colon tissue were, respectively, 3.2-fold, 3.5-fold, 2.2-fold and 1.6-fold higher than that using free 9-AC. In plasma, the high and sharp drug concentration profile from free drug was in contrast to the relatively low and flat pharmacokinetic profile obtained from drug released from the HPMA copolymer. There was no significant difference between released and free drug for the area under the concentration-time curve (AUC) and bioavailability values. As a consequence of the colon-specific release of unmodified 9-AC from the polymer conjugate, antitumor efficacy can be anticipated to be enhanced due to prolonged colon tumor exposure to higher and more localized drug concentrations.
Subject(s)
Camptothecin/analogs & derivatives , Drug Delivery Systems/methods , Methacrylates/administration & dosage , Administration, Oral , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Area Under Curve , Camptothecin/administration & dosage , Camptothecin/chemistry , Camptothecin/pharmacokinetics , Cecum/metabolism , Colon/metabolism , Feces/chemistry , Female , Injections, Intravenous , Intestine, Small/metabolism , Methacrylates/chemistry , Methacrylates/pharmacokinetics , Mice , Molecular Structure , Polymers/administration & dosage , Polymers/chemistry , Polymers/pharmacokinetics , Tissue DistributionABSTRACT
N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-9-aminocamptothecin (9-AC) conjugate for oral colon-specific drug delivery was designed, synthesized, and characterized. The drug, 9-AC, was attached to the polymer carrier via a spacer containing a combination of an aromatic azo bond and a 4-aminobenzylcarbamate group. The design of the spacer ensured a fast and highly efficient release of unmodified 9-AC from the polymer in the colon by azo bond cleavage followed by a 1,6-elimination mechanism. An in vitro degradation study indicated that this conjugate was stable in simulated upper GI tract conditions, including small intestine (SI) contents, SI mucosa suspension, and in PBS (pH 1.5 and 7.4). A fast release of the unmodified drug (85+/-10% of 9-AC in 12 h) was detected in rat cecal contents. This drug delivery system has potential in the treatment of colon cancer.