in-vivo non-viral delivery of shrna for mi

Delivery is the problem? Not at all! We in shRNAi land have many proven and effective methods of delivery. It's only the siRNA boys that struggle with this. I'm on a roll tonight, and presenting a great little IN VIVO study that not only demonstrates a ddRNAi way of recruiting stem cells to treat heart attacks (without Mesoblast's methods), is in the September 2011 edition of the premier journal "Circulation" of the American Heart Association and also demonstrates the "Non-viral minicircle" ddRNAi delivery system, that eliminates immune responses you may have with viral vectors as well as being very efficient and effective.

Circulation. 2011 Sep 13;124(11 Suppl):S46-54.
Double knockdown of prolyl hydroxylase and factor-inhibiting hypoxia-inducible factor with nonviral minicircle gene therapy enhances stem cell mobilization and angiogenesis after myocardial infarction.
Huang M, Nguyen P, Jia F, Hu S, Gong Y, de Almeida PE, Wang L, Nag D, Kay MA, Giaccia AJ, Robbins RC, Wu JC.
Source

Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA 94305-5454, USA.
Abstract
BACKGROUND:

Under normoxic conditions, hypoxia-inducible factor (HIF)-1a is rapidly degraded by 2 hydroxylases: prolyl hydroxylase (PHD) and factor-inhibiting HIF-1 (FIH). Because HIF-1a mediates the cardioprotective response to ischemic injury, its upregulation may be an effective therapeutic option for ischemic heart failure.
METHODS AND RESULTS:

PHD and FIH were cloned from mouse embryonic stem cells. The best candidate short hairpin (sh) sequences for inhibiting PHD isoenzyme 2 and FIH were inserted into novel, nonviral, minicircle vectors. In vitro studies after cell transfection of mouse C2C12 myoblasts, HL-1 atrial myocytes, and c-kit(+) cardiac progenitor cells demonstrated higher expression of angiogenesis factors in the double-knockdown group compared with the single-knockdown and short hairpin scramble control groups. To confirm in vitro data, shRNA minicircle vectors were injected intramyocardially after left anterior descending coronary artery ligation in adult FVB mice (n=60). Functional studies using MRI, echocardiography, and pressure-volume loops showed greater improvement in cardiac function in the double-knockdown group. To assess mechanisms of this functional recovery, we performed a cell trafficking experiment, which demonstrated significantly greater recruitment of bone marrow cells to the ischemic myocardium in the double-knockdown group. Fluorescence-activated cell sorting showed significantly higher activation of endogenous c-kit(+) cardiac progenitor cells. Immunostaining showed increased neovascularization and decreased apoptosis in areas of injured myocardium. Finally, western blots and laser-capture microdissection analysis confirmed upregulation of HIF-1a protein and angiogenesis genes, respectively.
CONCLUSIONS:

We demonstrated that HIF-1a upregulation by double knockdown of PHD and FIH synergistically increases stem cell mobilization and myocardial angiogenesis, leading to improved cardiac function.

PMID:
21911818
[PubMed - indexed for MEDLINE]
PMCID: PMC3181087
[Available on 2012/9/13]

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