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Human skin substitutes expressing il-12

a technology of human skin and il-12, which is applied in the field of human skin substitutes engineered to express exogenous il12, can solve the problems of reduced local tumor recurrence rate and treatment failure, and achieve the effect of preventing or inhibiting the spread of skin cancer

Inactive Publication Date: 2010-12-30
STRATATECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012]In some embodiments, the present invention provides methods of treating a patient comprising a) providing a human skin substitute comprising cells expressing heterologous Interleukin-12, wherein cells are selected from the group consisting of primary keratinocytes, keratinocyte precursors, transdifferentiated keratinocytes, and immortalized keratinocytes; and b) contacting said patient with said human skin substitute. In some embodiments, the patient has skin cancer. In some embodiments, the skin cancer is selected from the group consisting of melanoma and basal cell carcinoma. In some embodiments, the human skin substitute is applied to a site on said patient where a skin cancer has been removed. In some embodiments, the human skin substitute prevents or inhibits the spread of said skin cancer. In some embodiments, the human skin substitute produces amounts of bioactive IL-12 which are capable of inducing the proliferation of the PHA-stimulated lymphoblasts.

Problems solved by technology

Although surgical excision of the primary tumor is curative for the majority of squamous and basal cell skin cancers, approximately 10% of primary or recurrent non-melanoma skin cancers present as complex, perineural, or locally invasive tumors that are more difficult to manage by surgery alone.
The use of Mohs' micrographic surgery to eliminate the tumor and adjacent areas of microscopically abnormal tissue has led to decreases in the rate of local tumor recurrence.
However, tumor recurrence near the site of the primary tumor remains a predominant cause of treatment failure in these patients.

Method used

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  • Human skin substitutes expressing il-12
  • Human skin substitutes expressing il-12
  • Human skin substitutes expressing il-12

Examples

Experimental program
Comparison scheme
Effect test

example 1

NIKS® Skin Substitutes do not Express Detectable Levels of IL-12

[0086]Several groups have reported expression of IL-12 in cultured human keratinocytes as detected by sensitive nested RT-RCR methods [52-54]. However, analysis of IL-12 expression in organotypic skin cultures has not been reported. To determine the baseline level of IL-12 produced by skin substitutes prepared from NIKS® cells, a commercially-available IL-12 ELISA kit (R&D Systems, Minneapolis, Minn.) was used to quantify IL-12 in conditioned medium. Although the standard curve for the ELISA conformed to the kit validity criteria, IL-12 was not detected in conditioned medium at levels above the lowest IL-12 standard (31 pg / ml). These results demonstrate that skin substitute tissue prepared from NIKS® keratinocytes expresses little, if any IL-12.

example 2

IL-12 Vector Design and Construction

[0087]IL-12 is a heterodimer consisting of disulfide linked p40 and p35 subunits. Because the p40 subunit can form homodimers that are antagonistic to IL-12 bioactivity, it is important to avoid excess p40 production that could lead to the production of inhibitory p40 homodimers. To ensure expression of biologically active IL-12, the p40 and p35 subunits have been expressed as a fusion protein (p40 / p35) with the two separated by a short, flexible linker (Gly6Ser). Lieschke et al. demonstrated that this p40 / p35 fusion protein exhibited bioactivity comparable to recombinant human IL-12 (Lieschke et al., Nat Biotechnol., 15(1): 35-40 (1997)). This approach was shown to produce more bioactive IL-12 than co-transfection of cells with separate p35 and p40 expression constructs or bicistronic expression constructs where p35 and p40 were linked by an IRES.

[0088]The strategy for production of the DNA fragment encoding the p40 / p35 fusion protein is shown in...

example 3

Transient Transfection of NIKS® Cells in Monolayer Culture and Evaluation of IL-12 Fusion mRNA Levels

[0090]Expression of the IL-12 fusion protein have been evaluated initially in transiently-transfected cells. NIKS® cells were transfected with the IL-12 expression constructs or empty expression vectors. All transfected cultures were assayed for mRNA expression levels approximately 24 hrs post-transfection to verify transgene expression. Total RNA was isolated with Trizol (Invitrogen, Carlsbad Calif.) and treated with DNAse to eliminate residual transfected vector DNA. RT-PCR was performed using primers that specifically amplify the IL-12 p40 / p35 RNA transcribed from the expression constructs, but not endogenous IL-12 mRNA. The primers were designed to span an intron in the rabbit β-globin fragment in the vector such that products derived from mRNA are easily distinguished from products resulting from amplification of residual vector DNA. Culture medium from cells transiently-transfe...

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Abstract

The present invention relates generally to compositions for treating patients that have skin cancer or have recently had skin cancers removed. More specifically, the present invention provides human skin substitutes engineered to express exogenous IL-12 and compositions and methods for making human skin substitutes engineered to express exogenous IL-12. In addition, the present invention provides methods for treatment of sites on a patient where skin cancers have been removed with human skin substitutes engineered to express exogenous IL-12.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Prov. Appl. No. 61 / 180,250, filed May 21, 2009, the entire contents of which are incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]This Application was supported in part by the SBIR under Grant R44CA139747. The government has certain rights in the invention.FIELD OF THE INVENTION[0003]The present invention relates generally to compositions for treating patients that have skin cancer or have recently had skin cancers removed. More specifically, the present invention provides human skin substitutes engineered to express exogenous IL-12 and compositions and methods for making human skin substitutes engineered to express exogenous IL-12. In addition, the present invention provides methods for treatment of sites on a patient where skin cancers have been removed with human skin substitutes engineered to express exogenous IL-12.BACKGROUND[0004]Over ha...

Claims

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Application Information

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IPC IPC(8): A61K35/12A61P35/00C12N5/071C12N15/85
CPCA61K35/12A61K38/208A61K48/00C12N2510/00C12N5/0629C12N2501/23C07K14/5434A61P17/00A61P35/00
Inventor COMER, ALLEN R.ALLEN-HOFFMANN, B. LYNN
Owner STRATATECH
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