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Methods, compositions and apparatus for delivering heterologous molecules to cells

a technology of heterologous molecules and compositions, applied in the direction of genetic material ingredients, diagnostic recording/measuring, catheters, etc., can solve the problems of avoiding systemic delivery of therapeutic substances, minimizing the usefulness of such methods, and four essential components of the protocol listed above have stymied clinical translation, so as to avoid the risks of histamine, papaverine, heparin, and arterial access

Inactive Publication Date: 2007-03-29
THE TRUSTEES OF THE UNIV OF PENNSYLVANIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008] The present invention provides the first protocol for somatic gene transfer to muscle that achieves scale-independent, limb-wide transduction of nearly 100% of fibers, while avoiding the risks of histamine, papaverine, heparin, and arterial access.

Problems solved by technology

However, the four essential components of the protocol listed above have stymied clinical translation because of the inherent risk of hemorrhage, hypotension, and pulmonary dysfunction.
However, such agents can be associated with undesirable side effects, including short-term toxicity, which minimizes the usefulness of such methods.
However, these methods and devices avoid systemic delivery of therapeutic substances.
Further, studies in large animal models have revealed a trade-off between the efficiency of gene transfer using known methods and the inherent safety of the required pharmacological interventions.

Method used

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  • Methods, compositions and apparatus for delivering heterologous molecules to cells
  • Methods, compositions and apparatus for delivering heterologous molecules to cells
  • Methods, compositions and apparatus for delivering heterologous molecules to cells

Examples

Experimental program
Comparison scheme
Effect test

example 1

Evans Blue Dye (EBD) Study

[0115] EBD solution was prepared as described in C. R. Bridges, et al, Ann Thorac Surg, 73(6):1939-1946 (June 2002). A male adult Fisher rat underwent hindlimb isolation as described below.

[0116] Approximately 0.05 mg / g body weight of EBD was delivered in a sterile PBS & albumin solution. O.16 ml of the solution was diluted to a volume of 5 ml and infused through the greater saphenous vein with a 400 torr applied pressure into the isolated hindlimb. 2.4 ml of the solution was infused slowly into the contralateral hindlimb without isolation. The rat was necropsied 30 minutes after the start of infusion.

example 2

Materials

[0117] The following materials were used in the studies described in Examples 3 and 4, which demonstrate the method in a rat and canine model, respectively.

[0118] A. rAAV and rAd

[0119] All vector was procured through University of Pennsylvania Vector Core and was tested to be replication defective and endotoxin negative.

[0120] All vector used encoded the marker gene lac-Z coupled to a CMV promoter. Varying lots of vector were used for experiments with the goal of delivering ˜1013 genome copies (GC) / kg of rAAV and 1012 particles / kg of rAd5 in isolated limb infusion studies. The isolated hemibody trials delivered ˜5×1013 GC / kg of rAAV. rAAV serotype 2 / 1 was utilized for all experiments except for preliminary studies in the rat using rAAV serotype 2 / 2. The rAAV 2 / 1 used in the dogs was an aggregate pool of 5 lots.

[0121] B. Tissue Analysis

[0122] Tissue samples were ‘snap frozen’ in isopentane cooled in liquid nitrogen at the time of necropsy. 8 micron sections were genera...

example 3

Gene Transfer in Rat Model

[0123] A. Intravascular Injection of rAAV in Rat Isolated Limbs

[0124] After anesthesia with xylene and ketamine, inbred (male adult Fisher 344) rats underwent femoral artery and vein isolation with transmuscular placement of two overlapping 2-0 prolene tourniquets at the level of the proximal thigh. Through a small incision, the greater saphenous vein was cannulated with heat-tapered polyethylene tubes (PE 10; Becton Dickinson, Sparks, Md.) attached at their other ends to 30-gauge needles was performed. After tourniquets were tightened and the limb circulation was isolated, a total volume of 5 ml of sterile PBS and 1012 GC of rAAV encoding the lac-z marker gene was infused at pressures of 100, 200, 400 torr. The infusion time varied with delivery pressure, but the total time the rAAV solution was left in place was 30 minutes from the beginning of infusion. The tourniquets were then released and the catheter removed. The saphenous vein was ligated with a s...

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Abstract

A method for transferring a macromolecular complex to muscle cells by exsanguinating a region of the subject's microvasculature and delivering the complex to this region under high hydrostatic pressure is described. A balloon catheter having a balloon that extends substantially the full length of the cannula that is inserted into the subject is provided for use in the systemic delivery of macromolecular complex.

Description

BACKGROUND OF THE INVENTION [0001] The invention relates generally to the field of gene therapy. [0002] A variety of methods have been described in the literature as being useful for delivering a desired molecule into a target cell. The combined use of 1) complete vascular isolation using tourniquets and proximal arterio-venous cannulation, 2) systemic heparinization to prevent thrombosis, 3) peripheral vasodilation to optimize perfusion of muscle capillaries, and 4) histamine to produce physical gaps between adjacent endothelial cells has achieved widespread, homogeneous, vector-independent gene transfer to muscles of entire extremities. These studies began with marker genes and were then applied to several disease models in larger rodents and dogs. However, the four essential components of the protocol listed above have stymied clinical translation because of the inherent risk of hemorrhage, hypotension, and pulmonary dysfunction. [0003] For example, U.S. Pat. No. 6,177,403 descri...

Claims

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Application Information

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IPC IPC(8): A61M31/00A61F2/958A61K48/00A61MA61M29/00
CPCA61K48/0075A61M25/10A61M2025/1059A61M2025/1052A61M2025/105
Inventor STEDMAN, HANSELLHBRIDGES, CHARLES R.
Owner THE TRUSTEES OF THE UNIV OF PENNSYLVANIA
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