Synthegen Systems, Inc.

Beth Israel Deaconess Medical Center, Inc.

Zaheed Husain

Madhu Sudan Malo

Vikas P Sukhatme

Methods for treating cancer, e.g., in conjunction with anti-cancer therapy, like immunotherapy, and for identifying candidate therapeutic agents, by targeting ICAM4. While MDSCs in mice have been extensively characterized, their human counterparts are not well defined, and cell markers present in mice are not always usable in humans. MDSCs have been described as a heterogenous population of myeloid derived cells with immune suppressive capacity (5, 9, 40, 41). Recent renewed interest in the role of MDSC accumulation in human tumors has resulted in the increased need to define these cells better in order to target them for therapeutic intervention.

Cancer therapy targeting intercellular adhesion molecule 4 (ICAM4)

The present invention relates to the development of a positive selection vector based on insertional reconstruction of a reporter gene or of a regulatory gene controlling the expression of a reporter gene. The cloning vector carries a reporter gene or a regulatory gene with a mutation rendering the reporter or the regulatory gene protein functionally inactive. A primer carrying a nucleic acid sequence that corrects the mutation is used during PCR amplification of a targeted nucleic acid sequence, and the amplified DNA fragment is then ligated to the said vector thus reconstructing the wild-type reporter or regulatory gene.

pRGR: a positive selection vector system for direct cloning of PCR amplified DNA fragments

The present invention describes the development of a positive selection vector based on regulatory element modulation, wherein such modulation is achieved via insertional reconstruction or destruction of a regulatory element controlling transcription, translation, DNA replication and termination. A positive selection cloning vector pREM5Tc has been developed based on insertional reconstruction of a regulatory element of a reporter gene. The vector pREM5Tc carries the tetracycline resistance reporter gene with no functional &minus;35 region of its promoter, a regulatory element, thus resulting in no expression of the tetracycline resistance gene. Hence a host cell carrying the vector pREM5Tc is unable to produce the tetracycline resistance gene protein resulting in inhibition of its growth in presence of tetracycline. An consensus &minus;35 region is recognized as 5&prime;-TTGACA-3&prime; and a primer used in polymerase chain reaction (PCR) carries at its 5&prime; end the sequence 5&prime;-TGTCAA-3&prime;, which is the complementary sequence of 5&prime;-TTGACA-3&prime;. The PCR-amplified DNA fragment is ligated to pREM5Tc thus reconstructing the functional promoter of the tetracycline resistance reporter gene. Subsequent transformation of a host cell with the recombinant vector (carrying an insert DNA) results in production of the tetracycline resistance reporter gene protein that confers resistance to tetracycline thus allowing only the recombinants to grow in presence of tetracycline. The positive selection vector pREM5Tc greatly reduces, if not eliminates, the number of exonuclease-generated false positive clones.

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Medford, MA, United States of America

Zaheed Husain