Methods and compositions for nucleic acid transfer into cells

a technology of nucleic acids and compositions, applied in the direction of drug compositions, non-active genetic ingredients, genetic material ingredients, etc., can solve the problems that no one has specifically targeted or identified hdac6, and achieve the effect of increasing the fluid clearance in the lungs

Inactive Publication Date: 2009-03-12
NORTHWESTERN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010]In particular embodiments, the present invention provides methods for transfecting lung cells comprising: contacting lung cells with a gene transfer system and a vector under conditions of cyclic stretch such that gene expression from said vector is increased by employing said conditions of cyclic stretch compared to expression when cyclic stretch is not employed. In further embodiments, the contacting is in vivo in a subject. In some embodiments, the conditio

Problems solved by technology

While other studies have employed HDAC inhibitors to increase transcription, none have sp

Method used

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  • Methods and compositions for nucleic acid transfer into cells
  • Methods and compositions for nucleic acid transfer into cells
  • Methods and compositions for nucleic acid transfer into cells

Examples

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example 1

HDAC 6 Inhibition Results in Increase Transfection

[0062]In this Example, after establishing that acetylated microtubules are increased in stretched cells, and are still present in nocodazole treated cells, drug treatments were used to inhibit the tubulin deacetylase, HDAC6, and also HDAC6 knockdown cells, to increase the acetylation of microtubules. It was found that increased acetylation of microtubules increased gene transfer by increasing the trafficking of plasmids toward the nucleus. Finally, other targets of HDAC6 were examined and it was determined that they are not responsible for the observed increase in expression. Taken together, these results indicate that modulation of HDAC6 and the microtubule network can increase the efficiency of gene transfer.

Materials and Methods

Cell Culture Transfection, and Microinjection

[0063]Human adenocarcinoma A549 cells (ATCC, #CCL-185, Rockville, Md.) were grown in DMEM supplemented with 10% fetal bovine serum (FBS). A stable line of A549 c...

example 2

Tubulin Aceytlation and HDAC6 Activity in the Lung Under Cyclic Load

[0081]This Example investigates the relationship between acetylated microtubules and cyclic stretch. This Examples demonstrates that cyclic stretch, at parameters that mimic physiological respiration, increases the amount of acetylated microtubules both in vitro as well as in vivo. This Examples demonstrates that mechanical stimulation can alter tubulin post-translational modifications both in vitro as well as in vivo.

Materials and Methods

[0082]Cell Culture. All experiments, except where noted, were conducted on A549 cells, (ATCC, Manassas, Va.) a human lung adenocarcinoma cell line. Stably transfected A549 cells expressing siRNA against HDAC6 were a generous gift from T. P. Yao (Duke University) (27A) and were developed using a retrovirus system expressing an RNAi for HDAC6 causing a significant decrease in HDAC6 levels (33A). Cells were grown in high glucose DMEM supplemented with 10% fetal bovine serum, kanamyaci...

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Abstract

The present invention provides compositions and methods for increasing the transfer of nucleic acids into cells. In particular, the present invention provides for the use of inhibitors of HDAC6, a cytoplasmic histone deacetylase present in mammalian cells by, for example, small molecules or siRNA treatment, in increasing gene transfer and/or expression in cells in vitro and in vivo for research and gene therapy applications.

Description

[0001]The present application claims priority to U.S. Provisional Application 60 / 937,445, filed Jun. 27, 2007, which is herein incorporated by reference.[0002]This invention was made with government support under Grant No. HL71643, HL76139, and ESO13995 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE INVENTION[0003]The present invention provides compositions and methods for increasing the transfer of nucleic acids into cells. In particular, the present invention provides for the use of inhibitors of HDAC6, a cytoplasmic histone deacetylase present in mammalian cells by, for example, small molecules or siRNA treatment, in increasing gene transfer and / or expression in cells in vitro and in vivo for research and gene therapy applications.BACKGROUND[0004]A primary limitation of gene therapy, specifically non-viral gene therapy, is an inability to achieve high levels of expression. The cell and nuclear membranes as well as the...

Claims

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Application Information

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IPC IPC(8): A61K31/7088C12N15/09
CPCA61K31/7088A61K48/0008C12N2310/14C12N15/64C12N15/1137A61P43/00
Inventor DEAN, DAVID A.GEIGER, ROBERT CHRISTOPHER
Owner NORTHWESTERN UNIV
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