Systemic Vascular Transduction by Capsid Mutant Adeno-Associated Virus After Intravenous Injection

Lipinski, Daniel M.; Reid, Chris; Boye, Sanford L.; Peterson, Jim; Qi, Xiaoping; Boye, Shannon E.; Boulton, Michael E.; Hauswirth, William W.

doi:10.1089/hum.2015.097

Cited by 16 publications

(8 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Accordingly, several groups have attempted to achieve stable high-level transgene expression in EC. [3][4][5][6] Achievement of stable high-level transgene expression in EC requires a vector with significant endothelial tropism as well as a potent expression cassette that maintains activity indefinitely. We and others have shown that serotype 5 adenoviral vectors have high tropism for both animal and human EC.…”

Section: Introductionmentioning

confidence: 99%

StableIn VivoTransgene Expression in Endothelial Cells with Helper-Dependent Adenovirus: Roles of Promoter and Interleukin-10

Dronadula

Kwast

et al. 2017

Human Gene Therapy

View full text Add to dashboard Cite

Our long-term goal is to prevent or reverse atherosclerosis by delivering gene therapy from stably transduced endothelial cells (EC). We previously reported that EC-directed gene therapy with a helper-dependent adenovirus (HDAd) expressing apolipoprotein A-I (apo A-I) retarded development of atherosclerosis in rabbit carotid arteries over a 1-month interval. However, a 70% decline in apo A-I expression during this time raised concerns about long-term efficacy of this approach. Here we report use of several approaches aimed either at preventing this decline or at increasing apo A-I expression from HDAd at all time points: codon optimization, deletion of 3' untranslated sequences, substitution of a synthetic mammalian-based promoter (4XETE) for the cytomegalovirus (CMV) promoter, and co-transduction with an HDAd expressing interleukin-10. We tested these approaches using plasmid transfection of cultured EC and in vivo transduction of rabbit carotid artery EC. Codon optimization did not increase apo A-I expression. Deletion of 3' untranslated sequences extinguished apo A-I expression. Both substitution of 4XETE for the CMV promoter and expression of interleukin-10 stabilized apo A-I expression in vivo, although at the cost of lower early (3-day) expression levels. Surprisingly, both interventions stabilized apo A-I expression without altering the rate at which HDAd genomes were lost. These data establish that transgene expression from HDAd in EC is inherently stable in vivo and suggest that the early decline of CMV promoter-driven expression from HDAd-transduced EC is due neither to active downregulation of transcription nor to loss of HDAd genomes. Instead, apparent loss of expression from the CMV promoter appears to be a consequence of early (3-day) upregulation of CMV promoter activity via inflammatory pathways. Our results yield new paradigms to explain the early loss of genomes and transgene expression after in vivo gene transfer. These new paradigms will redirect strategies for achieving high-level, stable expression of transgenes in EC.

show abstract

Section: Introductionmentioning

confidence: 99%

StableIn VivoTransgene Expression in Endothelial Cells with Helper-Dependent Adenovirus: Roles of Promoter and Interleukin-10

Dronadula

Kwast

et al. 2017

Human Gene Therapy

View full text Add to dashboard Cite

show abstract

“… 42 Supporting this theory, in a previous study we demonstrated that rAAV2/2[QuadYF+TV] was neutralized by serum containing antibodies raised against AAV2. 43 …”

Section: Discussionmentioning

confidence: 99%

Improvement of Photoreceptor Targeting via Intravitreal Delivery in Mouse and Human Retina Using Combinatory rAAV2 Capsid Mutant Vectors

Reid

Ertel

Lipinski

2017

Invest. Ophthalmol. Vis. Sci.

Self Cite

View full text Add to dashboard Cite

PurposeEffective intravitreal gene delivery to cells of the central retina (i.e., photoreceptors) would be of substantial benefit for treating patients with retinal diseases, such as achromatopsia, where retinal detachment from a subretinal may be harmful. Previous studies demonstrated that mutation of the recombinant adeno-associated virus (rAAV) capsid through introduction of peptide insertions or amino acid substitutions dramatically alters vector tropism. Herein, we evaluate the photoreceptor transduction efficiency of three rAAV2/2-based capsid mutant vectors: rAAV2/2[7m8], rAAV2/2[QuadYF+TV], and a chimeric vector incorporating both mutations (termed rAAV2/2[MAX]) following intravitreal delivery in mice. Furthermore, we evaluate the transduction efficiency of rAAV2/2[MAX] using explanted human central retinal samples to address clinical translatability.MethodsVectors containing a GFP or mCherry reporter gene were intravitreally injected into C57BL/6J or Nrl-EGFP mice, respectively. Transduction was assessed in vivo utilizing a custom multiline confocal scanning laser ophthalmoscope. Injected Nrl-EGFP mouse retinas were used to quantify transduced photoreceptors using flow cytometry. Postmortem human retinal tissue was cultured following administration of rAAV2/2[MAX]. C57BL/6J retinas and human explants were cryosectioned to determine vector tropism.ResultsThe chimeric vector rAAV2/2[MAX] transduced significantly higher proportions of the retina than did either single mutant serotypes following intravitreal delivery in murine retina, including inner retinal cells and photoreceptors. Vector rAAV2[MAX] demonstrated transduction of human photoreceptors and ganglion cells.ConclusionsTransduction observed via rAAV2/2[MAX] indicates that combining mutations with complementary mechanisms of action in a single vector results in enhanced transduction. rAAV2/2[MAX] also presented the ability to transduce human photoreceptors and ganglion cells, indicating potential for efficient intravitreal vector delivery.

show abstract

“…AAV-N1-ICD and AAV control were generated by VectorBuilder, which include the endothelial-specific AAV2-QuadYF vectors 18 that encoded an intracellular fragment of mouse Notch1 (1749-2293aa, lacking the C-terminal PEST domain), 19 a T2A selfcleaving peptide sequence, and enhanced green fluorescent protein (EGFP) under the control of endothelial-specific Tie1 gene promoter (AAV2-QuadYF-mN1ICD-EGFP) or control vectors carrying the CMV promoter and EGFP (AAV2-QuadYF-EGFP). Mice (C57, 6-8 weeks) were anesthetized and injected retro-orbitally at a dose of 2×10 12 vg/mice in a total volume of 100 μL.…”

Section: Administration Of Aav-n1-icd and Aav Control In Ph Mouse Modelsmentioning

confidence: 99%

Reduced Notch1 Cleavage Promotes the Development of Pulmonary Hypertension

et al. 2022

View full text Add to dashboard Cite

Clinical trials of Dll4 (Delta-like 4) neutralizing antibodies (Dll4nAbs) in cancer patients are ongoing. Surprisingly, pulmonary hypertension (PH) occurs in 14% to 18% of patients treated with Dll4nAbs, but the mechanisms have not been studied. Here, PH progression was measured in mice treated with Dll4nAbs. We detected Notch signaling in lung tissues and analyzed pulmonary vascular permeability and inflammation. Notch target gene array was performed on adult human pulmonary microvascular endothelial cells (ECs) after inhibiting Notch cleavage. Similar mechanisms were studied in PH mouse models and pulmonary arterial hypertension patients. The rescue effects of constitutively activated Notch1 in vivo were also measured. We observed that Dll4nAbs induced PH in mice as indicated by significantly increased right ventricular systolic pressure, as well as pulmonary vascular and right ventricular remodeling. Mechanistically, Dll4nAbs inhibited Notch1 cleavage and subsequently impaired lung endothelial barrier function and increased immune cell infiltration in vessel walls. In vitro, Notch targeted genes’ expression related to cell growth and inflammation was decreased in human pulmonary microvascular ECs after the Notch1 inactivation. In lungs of PH mouse models and pulmonary arterial hypertension patients, Notch1 cleavage was inhibited. Consistently, EC cell-cell junction was leaky, and immune cell infiltration increased in PH mouse models. Overexpression activated Notch1-attenuated progression of PH in mice. In conclusion, Dll4nAbs led to PH development in mice by impaired EC barrier function and increased immune cell infiltration through inhibition of Notch1 cleavage in lung ECs. Reduced Notch1 cleavage in lung ECs could be an underlying mechanism of PH pathogenesis.

show abstract

Systemic Vascular Transduction by Capsid Mutant Adeno-Associated Virus After Intravenous Injection

Cited by 16 publications

References 47 publications

StableIn VivoTransgene Expression in Endothelial Cells with Helper-Dependent Adenovirus: Roles of Promoter and Interleukin-10

StableIn VivoTransgene Expression in Endothelial Cells with Helper-Dependent Adenovirus: Roles of Promoter and Interleukin-10

Improvement of Photoreceptor Targeting via Intravitreal Delivery in Mouse and Human Retina Using Combinatory rAAV2 Capsid Mutant Vectors

Reduced Notch1 Cleavage Promotes the Development of Pulmonary Hypertension

Contact Info

Product

Resources

About