Biomedical subjects

Alberto Auricchio

Publications and source records attributed to Alberto Auricchio.

At least 19 recordsLinked to original sources

PubMed2026

Targeted insertion of an optimized donor DNA is effective in a humanized mouse model of dominant retinitis pigmentosa.

Retinitis pigmentosa (RP) affects 1 in 3,000 individuals worldwide, with 30%-40% of cases inherited as autosomal dominant (AD). Mutations in RHO (RP4) are the most common cause of ADRP. Because most RHO mutations exert gain-of-function or dominant-negative effects, conventional gene supplementation is insufficient, requiring mutant allele inactivation. Allele-specific editing is impractical, as each mutation requires a unique therapeutic strategy. We present a mutation-agnostic, RHO-specific approach using adeno-associated viral vector-mediated homology-independent targeted integration (AAV-HITI). Optimized donor DNA design enables targeted integration and efficient transgene expression from the endogenous RHO locus. In a humanized RP4 mouse model harboring the RHO P23H mutant allele alongside an endogenous wild-type mouse Rho allele, AAV-HITI significantly improves retinal structure, function, and visual acuity up to 1 year post-treatment. Comprehensive molecular analyses characterize on-target editing in mouse retina and off-target editing in a human cell line. These findings establish an effective, human-centric AAV-HITI platform for RP4 and support its evaluation in this and other dominant genetic conditions.

AAV↗

PubMed2007

SUMF1 enhances sulfatase activities in vivo in five sulfatase deficiencies.

Sulfatases are enzymes that hydrolyse a diverse range of sulfate esters. Deficiency of lysosomal sulfatases leads to human diseases characterized by the accumulation of either GAGs (glycosaminoglycans) or sulfolipids. The catalytic activity of sulfatases resides in a unique formylglycine residue in their active site generated by the post-translational modification of a highly conserved cysteine residue. This modification is performed by SUMF1 (sulfatase-modifying factor 1), which is an essential factor for sulfatase activities. Mutations in the SUMF1 gene cause MSD (multiple sulfatase deficiency), an autosomal recessive disease in which the activities of all sulfatases are profoundly reduced. In previous studies, we have shown that SUMF1 has an enhancing effect on sulfatase activity when co-expressed with sulfatase genes in COS-7 cells. In the present study, we demonstrate that SUMF1 displays an enhancing effect on sulfatases activity when co-delivered with a sulfatase cDNA via AAV (adeno-associated virus) and LV (lentivirus) vectors in cells from individuals affected by five different diseases owing to sulfatase deficiencies or from murine models of the same diseases [i.e. MLD (metachromatic leukodystrophy), CDPX (X-linked dominant chondrodysplasia punctata) and MPS (mucopolysaccharidosis) II, IIIA and VI]. The SUMF1-enhancing effect on sulfatase activity resulted in an improved clearance of the intracellular GAG or sulfolipid accumulation. Moreover, we demonstrate that the SUMF1-enhancing effect is also present in vivo after AAV-mediated delivery of the sulfamidase gene to the muscle of MPSIIIA mice, resulting in a more efficient rescue of the phenotype. These results indicate that co-delivery of SUMF1 may enhance the efficacy of gene therapy in several sulfatase deficiencies.

Adenoviridae↗

PubMed2006

Preferential silencing of a common dominant rhodopsin mutation does not inhibit retinal degeneration in a transgenic model.

Autosomal dominant retinitis pigmentosa caused by the frequent rhodopsin P23H mutation is characterized by progressive photoreceptor cell death eventually leading to blindness and for which no therapies are available. Considering the gain-of-function effect exerted by the P23H mutation, strategies aimed at silencing the expression of the mutated allele, like RNA interference, are desirable. We have designed small interfering RNAs (siRNA) to silence specifically the P23H rhodopsin allele expressed by a transgenic rat model of the disease. We have selected in vitro one siRNA and generated an adeno-associated viral (AAV) vector expressing the short hairpin RNA (shRNA) based on the selected siRNA. In vitro the shRNA significantly inhibits the expression of the P23H but not the wild-type rhodopsin allele. Subretinal administration of the AAV2/5 vector encoding the shRNA in P23H transgenic rats results in inhibition of rhodopsin P23H expression that is not able to prevent or block photoreceptor degeneration. Since rhodopsin is the most abundant rod photoreceptor protein, systems resulting in more robust shRNA expression in the retina may be required to achieve therapeutic efficacy in vivo.

Alleles↗

PubMed2006

Correction of Hunter syndrome in the MPSII mouse model by AAV2/8-mediated gene delivery.

Mucopolysaccharidosis type II (MPSII; Hunter syndrome) is a lysosomal storage disorder caused by a deficiency in the enzyme iduronate 2-sulfatase (IDS). At present, the therapeutic approaches for MPSII are enzyme replacement therapy and bone marrow transplantation, although these therapies have some limitations. The availability of new AAV serotypes that display tissue-specific tropism and promote sustained expression of transgenes offers the possibility of AAV-mediated gene therapy for the systemic treatment of lysosomal diseases, including MPSII. We have characterized in detail the phenotype of IDS-deficient mice, a model of human MPSII. These mice display a progressive accumulation of glycosaminoglycans (GAGs) in many organs and excessive excretion of these compounds in their urine. Furthermore, they develop skeleton deformities, particularly of the craniofacial bones, and alopecia, they perform poorly in open-field tests and they have a severely compromised walking pattern. In addition, they present neuropathological defects. We have designed an efficient gene therapy approach for the treatment of these MPSII mice. AAV2/8TBG-IDS viral particles were administrated intravenously to adult MPSII mice. The plasma and tissue IDS activities were completely restored in all of the treated mice. This rescue of the enzymatic activity resulted in the full clearance of the accumulated GAGs in all of the tissues analyzed, the normalization of the GAG levels in the urine and the correction of the skeleton malformations. Overall, our findings suggest that this in vivo gene transfer approach has potential for the systemic treatment of patients with Hunter syndrome.

Animals↗

PubMed2006

Body-wide gene therapy of Duchenne muscular dystrophy in the mdx mouse model.

Duchenne muscular dystrophy is an X-linked muscle disease characterized by mutations in the dystrophin gene. Many of these can be corrected at the posttranscriptional level by skipping the mutated exon. We have obtained persistent exon skipping in mdx mice by tail vein injection with an adeno-associated viral (AAV) vector expressing antisense sequences as part of the stable cellular U1 small nuclear RNA. Systemic delivery of the AAV construct resulted in effective body-wide colonization, significant recovery of the functional properties in vivo, and lower creatine kinase serum levels, suggesting an overall decrease in muscle wasting. The transduced muscles rescued dystrophin expression and displayed a significant recovery of function toward the normal values at single muscle fiber level. This approach provides solid bases for a systemic use of AAV-mediated antisense-U1 small nuclear RNA expression for the therapeutic treatment of Duchenne muscular dystrophy.

Animals↗

PubMed2006

Aberrant splicing in the ocular albinism type 1 gene (OA1/GPR143) is corrected in vitro by morpholino antisense oligonucleotides.

An intronic point mutation was identified in the ocular albinism type 1 (OA1) gene (HUGO symbol, GPR143) in a family with the X-linked form of ocular albinism. Interestingly, the mutation creates a new acceptor splice site in intron 7 of the OA1 gene. In addition to low levels of normally spliced mRNA product of the OA1 gene, the patient samples contained also an aberrantly spliced mRNA with a 165 bp fragment of intron 7 (from position +750 to +914) inserted between exons 7 and 8. The abnormal transcript contained a premature stop codon and was unstable, as revealed by Northern blot analysis. We defined that mutation NC_000023.8:g.25288G>A generated a consensus binding motif for the splicing factor enhancer ASF/SF2, which most likely favored transcription of the aberrant mRNA. Furthermore, it activated a cryptic donor-splice site causing the inclusion between exons 7 and 8 of the 165 bp intronic fragment. Thus, the aberrant splicing is most likely explained by the generation of a de novo splicing enhancer motif. Finally, to rescue OA1 expression in the patient's melanocytes, we designed an antisense morpholino modified oligonucleotide complementary to the mutant sequence. The morpholino oligonucleotide (MO) was able to rescue OA1 expression and restore the OA1 protein level in the patient's melanocytes through skipping of the aberrant inclusion. The use of MO demonstrated that the lack of OA1 was caused by the generation of a new splice site. Furthermore, this technique will lead to new approaches to correct splice site mutations that cause human diseases.

Albinism, Ocular↗

PubMed2006

Chimeric adeno-associated virus/antisense U1 small nuclear RNA effectively rescues dystrophin synthesis and muscle function by local treatment of mdx mice.

Duchenne muscular dystrophy (DMD) is a X-linked myopathy in which deletions and point mutations in the dystrophin gene abolish dystrophin expression. The defect can often be corrected at the posttranscriptional level by exon skipping. In an animal model of DMD, the mdx mouse, a point mutation in exon 23 of the dystrophin gene introduces a premature stop codon. Skipping of this exon reestablishes the open reading frame in the dystrophin mRNA. We have obtained persistent exon skipping in mdx mice by local muscle injection of AAV vectors expressing antisense sequences fused to either U1 or U7 small nuclear RNA (snRNA). In the transduced muscles, dystrophin expression, amelioration of muscle morphology, and significant force recovery were obtained. These data indicate that the expression of antisense snRNAs, combined with their efficient muscular delivery through AAV vectors, is a powerful strategy for the therapeutic treatment of DMD. Like U7 snRNA, spliceosomal U1 snRNA is also a suitable backbone for the expression of antisense molecules active in exon skipping.

Animals↗

PubMed2006

AAV-mediated gene transfer for retinal diseases.

Vectors based on the adeno-associated virus (rAAV) are able to transduce the retina of animal models, including non-human primates, for a long-term period, safely and at sustained levels. The ability of the various rAAV serotypes to transduce retinal target cells has been exploited to successfully transfer genes to photoreceptors, retinal pigment epithelium and the inner retina, which are affected in many inherited and non-inherited blinding diseases. rAAV-mediated, constitutive and regulated gene expression at therapeutic levels has been achieved in the retina of animal models, thus providing proof-of-principle of gene therapy efficacy and safety in models of dominant and recessive retinal disorders. In addition, gene transfer of molecules with either neurotrophic or antiangiogenic properties provides useful alternatives to the classic gene replacement for treatment of both mendelian and complex traits affecting the retina. Years of successful rAAV-mediated gene transfer to the retina have resulted in restoration of vision in dogs affected with congenital blindness. This has paved the way to the first attempts at treating inherited retinal diseases in humans with rAAV. Although the results of rAAV clinical trials for non-retinal diseases give a warning that the outcome of viral-mediated gene transfer in humans may be different from that predicted based on results in other species, the immune privilege of the retina combined with the versatility of rAAV serotypes may ultimately provide the first successful treatment of human inherited diseases using rAAV.

Adenoviridae↗

PubMed2005

Inhibition of ocular neovascularization by hedgehog blockade.

Ocular neovascularization associated with proliferative diabetic retinopathy and age-related macular degeneration is the leading cause of severe visual loss in adults in developed countries. Physiological and pathological retinal angiogenesis may occur independently in postnatal life through the complex activation of pro- and antiangiogenic pathways. We report that the Sonic hedgehog (Shh) pathway is activated in the retina in animal models of retinal and choroidal neovascularization. We show that pharmacological inhibition of the Shh signaling pathway significantly reduces physiological retinal angiogenesis and inhibits pathological vascularization in both models. Under retinal hypoxic conditions, inhibition of the Shh pathway results in reduction of vascular endothelial growth factor (VEGF) level, along with that of Patched-1 (Ptch1), a canonical Shh target, thus placing Shh activation upstream of VEGF in experimental retinal neovascularization. Our data demonstrate the requirement of the Shh pathway for retinal angiogenesis and its inhibition as a potential therapeutic strategy targeting ocular neovascular disease.

Animals↗

PubMed2005

Intramuscular viral delivery of paraplegin rescues peripheral axonopathy in a model of hereditary spastic paraplegia.

Degeneration of peripheral motor axons is a common feature of several debilitating diseases including complicated forms of hereditary spastic paraplegia. One such form is caused by loss of the mitochondrial energy-dependent protease paraplegin. Paraplegin-deficient mice display a progressive degeneration in several axonal tracts, characterized by the accumulation of morphological abnormal mitochondria. We show that adenoassociated virus-mediated (AAV-mediated) intramuscular delivery of paraplegin halted the progression of neuropathological changes and rescued mitochondrial morphology in the peripheral nerves of paraplegin-deficient mice. One single injection before onset of symptoms improved the motor performance of paraplegin-deficient mice for up to 10 months, indicating that the peripheral neuropathy contributes to the clinical phenotype. This study provides a proof of principle that gene transfer may be an effective therapeutic option for patients with paraplegin deficiency and demonstrates that AAV vectors can be successfully employed for retrograde delivery of an intracellular protein to spinal motor neurons, opening new perspectives for several hereditary axonal neuropathies of the peripheral nerves.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2005

Natural antisense transcripts associated with genes involved in eye development.

Natural antisense transcripts (NATs) are a class of genes whose role in controlling gene expression is becoming more and more relevant. We describe the identification of eight novel mouse NATs associated with transcription factors (Pax6, Pax2, Six3, Six6, Otx2, Crx, Rax and Vax2) that play an important role in eye development and function. These newly identified NATs overlap with the mature processed mRNAs or with the primary unprocessed transcript of their corresponding sense genes, are predicted to represent either protein coding or non-coding RNAs and undergo extensive alternative splicing. Expression studies, by both RT-PCR and RNA in situ hybridization, demonstrate that most of these NATs, similarly to their sense counterparts, display a specific or predominant expression in the retina, particularly at postnatal stages. We found a significant reduction of the expression levels of one of these NATs, Vax2OS (Vax2 opposite strand) in a mouse mutant carrying the inactivation of Vax2, the corresponding sense gene. In addition, we overexpressed another NAT, CrxOS, in mouse adult retina using adeno-associated viral vectors and we observed a significant decrease in the expression levels of the corresponding sense gene, Crx. These results suggest that these transcripts are functionally related to their sense counterparts and may play an important role in regulating the molecular mechanisms that underlie eye development and function in both physiological and pathological conditions.

Alternative Splicing↗

PubMed2005

Amelioration of both functional and morphological abnormalities in the retina of a mouse model of ocular albinism following AAV-mediated gene transfer.

X-linked recessive ocular albinism type I (OA1) is due to mutations in the OA1 gene (approved gene symbol GPR143), which is expressed in the retinal pigment epithelium (RPE). The Oa1 (Gpr143) knockout mouse (Oa1(-/-)) model recapitulates many of the OA1 retinal morphological anomalies, including a lower number of melanosomes of increased size in the RPE. The Oa1(-/-) mouse also displays some of the retinal developmental abnormalities observed in albino patients such as misrouting of the optic tracts. Here, we show that these anomalies are associated with retinal electrophysiological abnormalities, including significant decrease in a- and b-wave amplitude and delayed recovery of b-wave amplitude from photoreceptor desensitization following bright light exposure. This suggests that lack of Oa1 in the RPE impacts on photoreceptor activity. More interestingly, adeno-associated viral vector-mediated Oa1 gene transfer to the retina of the Oa1(-/-) mouse model results in significant recovery of its retinal functional abnormalities. In addition, Oa1 retinal gene transfer increases the number of melanosomes in the Oa1(-/-) mouse RPE. Our data show that gene transfer to the adult retina unexpectedly rescues both functional and morphological abnormalities in a retinal developmental disorder, opening novel potential therapeutic perspectives for this and other forms of albinism.

Albinism, Ocular↗

PubMed2005

Long-term inducible gene expression in the eye via adeno-associated virus gene transfer in nonhuman primates.

Adeno-associated viral gene therapy has shown promise for the treatment of inherited and degenerative diseases in a variety of animal models. Some of the most dramatic results have been obtained in the field of ocular gene therapy, where efficacy has been tremendous in inherited and acquired retinal disorders. For the promise of this approach to be realized it will be necessary to create vectors capable of pharmacologic or physiologic regulation of the transgene. We describe in this paper a dimerizer-inducible viral expression system that is able to reproducibly drive expression of the reporter gene erythropoietin in the eyes of nonhuman primates over a period of 2.5 years. The expression profiles were characterized by minimal basal expression in the absence of inducer and dose-responsive maximal expression in the presence of inducer drug.

Animals↗

PubMed2005

Adeno-associated viral vectors for retinal gene transfer and treatment of retinal diseases.

Retinal gene transfer holds big promises for the treatment of inherited and non-inherited blinding diseases, such as retinitis pigmentosa or age-related macular degeneration. Key to the development of successful gene-based therapies for the eye are efficient tools for retinal gene transfer. Vectors based on adeno-associated viruses (AAV) are able to transduce robustly and persistently different retinal cell types of animal models after a single intraocular administration. Recombinant AAV (rAAV) vectors are versatile gene transfer tools in that capsid proteins from dozens of AAV serotypes can be easily interchanged, resulting in the creation of recombinant vectors with unique transduction properties. This has allowed successful proof-of-principle studies using rAAV-mediated gene transfer to restore retinal morphology and function in small and large animal models of retinal diseases. In addition, gene delivery using rAAV vectors in the eye seems to have appropriate biosafety characteristics to rapidly move it from bench to bedside. All the above aspects will be reviewed and discussed in detail below.

Angiogenesis Inhibitors↗

PubMed2005

Nonhuman primate models for diabetic ocular neovascularization using AAV2-mediated overexpression of vascular endothelial growth factor.

Neovascularization leads to blindness in numerous ocular diseases, including diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, and sickle cell disease. More effective and stable treatments for ocular neovascularization are needed, yet there are major limitations in the present animal models. To develop primate models of diabetic retinopathy and choroidal neovascularization, rhesus monkeys were injected subretinally or intravitreally with an adeno-associated virus (AAV)-2 vector carrying the cDNA encoding human vascular endothelial growth factor (VEGF). Overexpression of VEGF was measured by intraocular fluid sampling over time. Neovascularization was evaluated by ophthalmoscopy through angiography, optical coherence tomography, and ultimately histopathology. Overexpression of VEGF through AAV2 results in rapid development of features of diabetic retinopathy or macular edema, depending on the targeted cell type/mode of production of VEGF and diffusion of VEGF. Nonhuman primate models will be useful in testing long-term safety and efficacy of novel therapeutic agents for blinding neovascular diseases.

Adenoviridae↗

PubMed2004

Effect of over-expression of pigment epithelium derived factor (PEDF) on developing retinal vasculature in the mouse.

PURPOSE: Pigment epithelium derived factor (PEDF) is a secreted protein with demonstrated anti-angiogenic properties, and with potential application for the treatment of neovascular disease. Delivery of pigment epithelium derived factor to the retina via virus mediated gene transfer has been shown to inhibit neovascularization in a number of experimental models. While pigment epithelium derived factor is endogenously expressed in the retina, its role in guiding normal vessel development and growth is not yet known. This study aimed to determine whether over-expression of pigment epithelium derived factor alters the normal pattern of retinal vessel development. METHODS: Neonatal (age postnatal day 2 (P2)) CD1 mice were injected subretinally unilaterally with AAV2/1.CMV.PEDF while contralateral eyes were injected subretinally with AAV2/1.CMV.EGFP as control. Cohorts of animals were sacrificed at P7 to P21 and the retinal vasculature was co-labeled through fluorescein-dextran perfusion and immunohistochemistry. Vascular size, localization, and structure were analyzed using light and confocal microscopy. Additional cohorts were use to obtain quantitative levels of pigment epithelium derived factor protein through ELISA. RESULTS: The extent of vessel growth from the optic disk to periphery over time (i.e., the radius of retinal vasculature), and the area of expansion of the neural retina were unaffected by over-expression of pigment epithelium derived factor to levels at least 3.5 fold higher than endogenous levels. The thicknesses of the various retinal layers were similar in AAV2/1.CMV.PEDF treated and control injected eyes. Three dimensional analysis of confocal images shows a slight delay in the rate of growth of vasculature into the deeper layers of the retina in pigment epithelium derived factor treated eyes compared to EGFP treated control eyes. However, the normal differentiation of vessels into arterioles, and venules, and the formation of a capillary network continued to occur, achieving normal and complete maturation of vascular structure by P21. CONCLUSIONS: Over-expression of pigment epithelium derived factor in the developing retina exerted no marked or permanent effects on retinal vessel growth and differentiation. The findings are relevant to the safety of the potential therapeutic use of pigment epithelium derived factor in human retinal disease.

Animals↗

PubMed2004

Systemic but not intraocular Epo gene transfer protects the retina from light-and genetic-induced degeneration.

Molecules with neurotrophic activity are being evaluated for treatment of retinitis pigmentosa in animal models. In particular, great interest has been focused recently on erythropoietin (Epo). Evidence of its neurotrophic activity comes mainly from data demonstrating photoreceptor protection in a rodent light-damage model through systemic administration of a recombinant form of this hormone. Our goal was to test whether Epo retinal gene transfer can rescue or delay photoreceptor cell death. We delivered adeno-associated viral vectors encoding Epo intraocularly and, for comparison, intramuscularly to one light-induced and two genetic models of retinal degeneration. Intraocular Epo gene transfer resulted in sustained hormone expression in the eye, which was undetectable systemically. In contrast, Epo intramuscular gene transfer resulted in hormone secretion in the circulation, which was not detected in ocular fluids. The protein secreted from muscle and retina is of the same molecular weight as a commercial recombinant human Epo. Interestingly, following systemic but not intraocular Epo delivery, morphological photoreceptor protection was observed in the light-damage and rds/peripherin (Prph2) models of retinal degeneration. In the light-damage model, the morphological rescue was accompanied by a significant electrophysiological improvement of photoreceptor function. In contrast, no photoreceptor rescue was observed following Epo gene transfer in the rd10 model. This suggests that different apoptotic mechanisms, with varying sensitivities to Epo, occur in different retinal degeneration models. In conclusion, our data support Epo as a neuroprotective agent in some, but not all, retinal degenerations. Further, rescue is observed in specific models after systemic but not intraocular Epo gene transfer.

Animals↗

PubMed2004

Advances in AAV-mediated gene transfer for the treatment of inherited disorders.

The holy grail of gene therapy is the cure of genetic diseases. To achieve this goal, a vector system is desirable that offers a high level of safety combined with clinical efficacy and versatility in terms of potential applications. Gene therapy vectors based on recombinant adeno-associated viruses (AAVs) meet all of these criteria: They are nonpathogenic, devoid of viral coding sequences, and mediate long-term gene expression in the absence of an immune or inflammatory response. Moreover, with the recent discovery of novel AAV serotypes, there is now one preferred serotype for nearly every organ or tissue to target. Thus, AAV gene therapy vectors are increasingly becoming the vectors of choice for the treatment of inherited disorders.

Dependovirus↗