title: Excision of HIV-1 proviral DNA by recombinant cell permeable tre-recombinase.
abstract: Over the previous years, comprehensive studies on antiretroviral drugs resulted in the successful introduction of highly active antiretroviral therapy (HAART) into clinical practice for treatment of HIV/AIDS. However, there is still need for new therapeutic approaches, since HAART cannot eradicate HIV-1 from the infected organism and, unfortunately, can be associated with long-term toxicity and the development of drug resistance. In contrast, novel gene therapy strategies may have the potential to reverse the infection by eradicating HIV-1. For example, expression of long terminal repeat (LTR)-specific recombinase (Tre-recombinase) has been shown to result in chromosomal excision of proviral DNA and, in consequence, in the eradication of HIV-1 from infected cell cultures. However, the delivery of Tre-recombinase currently depends on the genetic manipulation of target cells, a process that is complicating such therapeutic approaches and, thus, might be undesirable in a clinical setting. In this report we demonstrate that E.coli expressed Tre-recombinases, tagged either with the protein transduction domain (PTD) from the HIV-1 Tat trans-activator or the translocation motif (TLM) of the Hepatitis B virus PreS2 protein, were able to translocate efficiently into cells and showed significant recombination activity on HIV-1 LTR sequences. Tre activity was observed using episomal and stable integrated reporter constructs in transfected HeLa cells. Furthermore, the TLM-tagged enzyme was able to excise the full-length proviral DNA from chromosomal integration sites of HIV-1-infected HeLa and CEM-SS cells. The presented data confirm Tre-recombinase activity on integrated HIV-1 and provide the basis for the non-genetic transient application of engineered recombinases, which may be a valuable component of future HIV eradication strategies.
authors.affiliation: Heinrich Pette Institute - Leibniz Institute for Experimental Virology, Hamburg, Germany.
authors.forename: Lakshmikanth
authors.initials: L
authors.lastname: Mariyanna
authors.forename: Poornima
authors.initials: P
authors.lastname: Priyadarshini
authors.forename: Helga
authors.initials: H
authors.lastname: Hofmann-Sieber
authors.forename: Marcel
authors.initials: M
authors.lastname: Krepstakies
authors.forename: Nicole
authors.initials: N
authors.lastname: Walz
authors.forename: Adam
authors.initials: A
authors.lastname: Grundhoff
authors.forename: Frank
authors.initials: F
authors.lastname: Buchholz
authors.forename: Eberhard
authors.initials: E
authors.lastname: Hildt
authors.forename: Joachim
authors.initials: J
authors.lastname: Hauber
mesh_terms: D002463:Cell Membrane Permeability; D003001:Cloning, Molecular; D004260:DNA Repair; D004279:DNA, Viral / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015658:HIV Infections / Q000628:therapy*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D011994:Recombinant Proteins / Q000627:therapeutic use; D045522:Recombinases / Q000008:administration & dosage* / Q000378:metabolism / Q000627:therapeutic use
chemical_list: D004279:DNA, Viral; D011994:Recombinant Proteins; D045522:Recombinases