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1.
Gene Ther ; 25(3): 205-219, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29785047

RESUMEN

The successful application of adeno-associated virus (AAV) gene delivery vectors as a therapeutic paradigm will require efficient gene delivery to the appropriate cells in affected organs. In this study, we utilized a rational design approach to introduce modifications to the AAV2 and AAVrh8R capsids and the resulting variants were evaluated for transduction activity in the retina and brain. The modifications disrupted either capsid/receptor binding or altered capsid surface charge. Specifically, we mutated AAV2 amino acids R585A and R588A, which are required for binding to its receptor, heparan sulfate proteoglycans, to generate a variant referred to as AAV2-HBKO. In contrast to parental AAV2, the AAV2-HBKO vector displayed low-transduction activity following intravitreal delivery to the mouse eye; however, following its subretinal delivery, AAV2-HBKO resulted in significantly greater photoreceptor transduction. Intrastriatal delivery of AAV2-HBKO to mice facilitated widespread striatal and cortical expression, in contrast to the restricted transduction pattern of the parental AAV2 vector. Furthermore, we found that altering the surface charge on the AAVrh8R capsid by modifying the number of arginine residues on the capsid surface had a profound impact on subretinal transduction. The data further validate the potential of capsid engineering to improve AAV gene therapy vectors for clinical applications.


Asunto(s)
Terapia Genética/métodos , Parvovirinae/crecimiento & desarrollo , Parvovirinae/inmunología , Animales , Encéfalo/metabolismo , Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Dependovirus/inmunología , Técnicas de Transferencia de Gen , Vectores Genéticos , Células HeLa , Heparitina Sulfato , Humanos , Ratones , Ratones Endogámicos C57BL , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Transducción Genética/métodos
2.
Mol Ther ; 18(11): 1983-94, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20736932

RESUMEN

Liver-directed gene therapy with adeno-associated virus (AAV) vectors effectively treats mouse models of lysosomal storage diseases (LSDs). We asked whether these results were likely to translate to patients. To understand to what extent preexisting anti-AAV8 antibodies could impede AAV8-mediated liver transduction in primates, commonly preexposed to AAV, we quantified the effects of preexisting antibodies on liver transduction and subsequent transgene expression in mouse and nonhuman primate (NHP) models. Using the highest viral dose previously reported in a clinical trial, passive transfer of NHP sera containing relatively low anti-AAV8 titers into mice blocked liver transduction, which could be partially overcome by increasing vector dose tenfold. Based on this and a survey of anti-AAV8 titers in 112 humans, we predict that high-dose systemic gene therapy would successfully transduce liver in >50% of human patients. However, although high-dose AAV8 administration to mice and monkeys with equivalent anti-AAV8 titers led to comparable liver vector copy numbers, the resulting transgene expression in primates was ~1.5-logs lower than mice. This suggests vector fate differs in these species and that strategies focused solely on overcoming preexisting vector-specific antibodies may be insufficient to achieve clinically meaningful expression levels of LSD genes using a liver-directed gene therapy approach in patients.


Asunto(s)
Dependovirus/genética , Terapia Genética , Hepatocitos/inmunología , Enfermedades por Almacenamiento Lisosomal/terapia , Transgenes/fisiología , alfa-Galactosidasa/sangre , Animales , Anticuerpos Neutralizantes/inmunología , Western Blotting , Vectores Genéticos/administración & dosificación , Células HeLa , Hepatocitos/metabolismo , Humanos , Enfermedades por Almacenamiento Lisosomal/genética , Enfermedades por Almacenamiento Lisosomal/inmunología , Macaca fascicularis , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Plasmaféresis , Biosíntesis de Proteínas , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , alfa-Galactosidasa/genética
3.
Nat Commun ; 12(1): 4219, 2021 07 09.
Artículo en Inglés | MEDLINE | ID: mdl-34244505

RESUMEN

Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a "GG" dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases.


Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Staphylococcus/enzimología , Animales , Proteína 9 Asociada a CRISPR/aislamiento & purificación , Línea Celular Tumoral , Dependovirus , Modelos Animales de Enfermedad , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Células HEK293 , Humanos , Macaca fascicularis , Masculino , Ratones , Parvovirinae/genética , Ingeniería de Proteínas , Ribonucleasas , Staphylococcus/genética , Especificidad por Sustrato , Síndromes de Usher/genética , Síndromes de Usher/terapia , ARN Guía de Sistemas CRISPR-Cas
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