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1.
Hepatol Commun ; 7(2): e0009, 2023 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-37074875

RESUMEN

BACKGROUND AND AIMS: Adeno-associated virus (AAV) vectors are widely used to deliver therapeutic transgenes to distinct tissues, including the liver. Vectors based on naturally occurring AAV serotypes as well as vectors using engineered capsids have shown variations in tissue tropism and level of transduction between different mouse models. Moreover, results obtained in rodents frequently lack translatability into large animal studies. In light of the increasing interest in AAV vectors for human gene therapy, an increasing number of studies are being performed in nonhuman primates. To keep animal numbers to a minimum and thus optimize the process of AAV capsid selection, we developed a multiplex barcoding approach to simultaneously evaluate the in vivo vector performance for a set of serotypes and capsid-engineered AAV vectors across multiple organs. APPROACH AND RESULTS: Vector biodistribution and transgene expression were assessed by quantitative PCR, quantitative reverse transcription PCR, vector DNA amplicon Illumina sequencing and vRNAseq in male and female rhesus macaques simultaneously dosed with a mixture of barcoded naturally occurring or engineered AAV vectors encoding the same transgene. As expected, our findings show animal-to-animal variation in both the biodistribution and tissue transduction pattern, which was partly influenced by each animal's distinctive serological status. CONCLUSIONS: This method offers a robust approach to AAV vector optimization that can be used to identify and validate AAV vectors for gene delivery to potentially any anatomical site or cell type.


Asunto(s)
Cápside , Dependovirus , Animales , Ratones , Femenino , Masculino , Humanos , Cápside/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Distribución Tisular , Macaca mulatta/genética , Macaca mulatta/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Terapia Genética/métodos
2.
Mol Ther Methods Clin Dev ; 20: 258-275, 2021 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-33473359

RESUMEN

Chronic hepatitis B virus (HBV) infection is a major public health problem. New treatment approaches are needed because current treatments do not target covalently closed circular DNA (cccDNA), the template for HBV replication, and rarely clear the virus. We harnessed adeno-associated virus (AAV) vectors and CRISPR-Staphylococcus aureus (Sa)Cas9 to edit the HBV genome in liver-humanized FRG mice chronically infected with HBV and receiving entecavir. Gene editing was detected in livers of five of eight HBV-specific AAV-SaCas9-treated mice, but not control mice, and mice with detectable HBV gene editing showed higher levels of SaCas9 delivery to HBV+ human hepatocytes than those without gene editing. HBV-specific AAV-SaCas9 therapy significantly improved survival of human hepatocytes, showed a trend toward decreasing total liver HBV DNA and cccDNA, and was well tolerated. This work provides evidence for the feasibility and safety of in vivo gene editing for chronic HBV infections, and it suggests that with further optimization, this approach may offer a plausible way to treat or even cure chronic HBV infections.

3.
Hum Gene Ther ; 32(1-2): 96-112, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32998579

RESUMEN

Adeno-associated virus (AAV) vectors such as AAV6, which shows tropism for primary human CD4+ T cells in vitro, are being explored for delivery of anti-HIV therapeutic modalities in vivo. However, pre-existing immunity and sequestration in nontarget organs can significantly hinder their performance. To overcome these challenges, we investigated whether immunosuppression would allow gene delivery by AAV6 or targeted AAV6 derivatives in seropositive rhesus macaques. Animals were immune suppressed with rapamycin before intravenous (IV) or subcutaneous (SC) delivery of AAV, and we monitored vector biodistribution, gene transfer, and safety. Macaques received phosphate-buffered saline, AAV6 alone, or an equal dose of AAV6 and an AAV6-55.2 vector retargeted to CD4 through a direct ankyrin repeat protein (DARPin). AAV6 and AAV6-55.2 vector genomes were found in peripheral blood mononuclear cells and most organs up to 28 days postadministration, with the highest levels seen in liver, spleen, lymph nodes (LNs), and muscle, suggesting that retargeting did not prevent vector sequestration. Despite vector genome detection, gene expression from AAV6-55.2 was not detected in any tissue. SC injection of AAV6 facilitated efficient gene expression in muscle adjacent to the injection site, plus low-level gene expression in spleen, LNs, and liver, whereas gene expression following IV injection of AAV6 was predominantly seen in the spleen. AAV vectors were well tolerated, although elevated liver enzymes were detected in three of four AAV-treated animals 14 days after rapamycin withdrawal. One SC-injected animal had muscle inflammation proximal to the injection site, plus detectable T cell responses against transgene and AAV6 capsid at study finish. Overall, our data suggest that rapamycin treatment may offer a possible strategy to express anti-HIV therapeutics such as broadly neutralizing antibodies from muscle. This study provides important safety and efficacy data that will aid study design for future anti-HIV gene therapies.


Asunto(s)
Dependovirus , Vectores Genéticos , Animales , Dependovirus/genética , Proteínas de Repetición de Anquirina Diseñadas , Vectores Genéticos/genética , Humanos , Leucocitos Mononucleares , Macaca mulatta , Sirolimus/uso terapéutico , Distribución Tisular
4.
Clin Sci (Lond) ; 130(8): 613-23, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26795436

RESUMEN

Perinatal growth restriction (GR) is associated with heightened sympathetic tone and hypertension. We have previously shown that naturally occurring neonatal GR programmes hypertension in male but not female mice. We therefore hypothesized that intact ovarian function or post-ovariectomy (OVX) oestrogen administration protects GR female mice from hypertension. Utilizing a non-interventional model that categorizes mice with weanling weights below the tenth percentile as GR, control and GR adult mice were studied at three distinct time points: baseline, post-OVX and post-OVX with oral oestrogen replacement. OVX elicited hypertension in GR mice that was significantly exacerbated by psychomotor arousal (systolic blood pressure at light to dark transition: control 122 ± 2; GR 119 ± 2; control-OVX 116 ± 3; GR-OVX 126 ± 3 mmHg). Oestrogen partially normalized the rising blood pressure surge seen in GR-OVX mice (23 ± 7% reduction). GR mice had left ventricular hypertrophy, and GR-OVX mice in particular had exaggerated bradycardic responses to sympathetic blockade. For GR mice, a baseline increase in baroreceptor reflex sensitivity and high frequency spectral power support a vagal compensatory mechanism, and that compensation was lost following OVX. For GR mice, the OVX-induced parasympathetic withdrawal was partially restored by oestrogen (40 ± 25% increase in high frequency spectral power, P<0.05). In conclusion, GR alters cardiac morphology and cardiovascular regulation. The haemodynamic consequences of GR are attenuated in ovarian-sufficient or oestrogen-replete females. Further investigations are needed to define the role of hormone replacement therapy targeted towards young women with oestrogen deficiency and additional cardiovascular risk factors, including perinatal GR, cardiac hypertrophy and morning surge hypertension.


Asunto(s)
Presión Sanguínea/efectos de los fármacos , Peso Corporal , Sistema Cardiovascular/efectos de los fármacos , Ritmo Circadiano , Estradiol/administración & dosificación , Terapia de Reemplazo de Estrógeno , Hipertensión/prevención & control , Ovariectomía , Adaptación Fisiológica , Administración Oral , Antagonistas de Receptores Adrenérgicos alfa 1/farmacología , Animales , Barorreflejo/efectos de los fármacos , Sistema Cardiovascular/inervación , Modelos Animales de Enfermedad , Femenino , Bloqueadores Ganglionares/farmacología , Frecuencia Cardíaca , Hipertensión/etiología , Hipertensión/fisiopatología , Ratones Endogámicos C57BL , Actividad Motora , Antagonistas Nicotínicos/farmacología , Factores de Tiempo
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