Helga Hofmann-Sieber

Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity

Current combination antiretroviral therapies (cART) efficiently suppress HIV-1 reproduction in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. To generate an antiviral agent capable of eradicating the provirus from infected cells, we employed 145 cycles of substrate-linked directed evolution to evolve a recombinase (Brec1) that site-specifically recognizes a 34-bp sequence present in the long terminal repeats (LTRs) of the majority of the clinically relevant HIV-1 strains and subtypes. Brec1 efficiently, precisely and safely removes the integrated provirus from infected cells and is efficacious on clinical HIV-1 isolates in vitro and in vivo, including in mice humanized with patient-derived cells. Our data suggest that Brec1 has potential for clinical application as a curative HIV-1 therapy.

Highly Significant Antiviral Activity of HIV-1 LTR-Specific Tre-Recombinase in Humanized Mice

Stable integration of HIV proviral DNA into host cell chromosomes, a hallmark and essential feature of the retroviral life cycle, establishes the infection permanently. Current antiretroviral combination drug therapy cannot cure HIV infection. However, expressing an engineered HIV-1 long terminal repeat (LTR) site-specific recombinase (Tre), shown to excise integrated proviral DNA in vitro, may provide a novel and highly promising antiviral strategy. We report here the conditional expression of Tre-recombinase from an advanced lentiviral self-inactivation (SIN) vector in HIV-infected cells. We demonstrate faithful transgene expression, resulting in accurate provirus excision in the absence of cytopathic effects. Moreover, pronounced Tre-mediated antiviral effects are demonstrated in vivo, particularly in humanized Rag2−/−γc−/− mice engrafted with either Tre-transduced primary CD4+ T cells, or Tre-transduced CD34+ hematopoietic stem and progenitor cells (HSC). Taken together, our data support the use of Tre-recombinase in novel therapy strategies aiming to provide a cure for HIV.

Excision of HIV-1 Proviral DNA by Recombinant Cell Permeable Tre-Recombinase

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.

Engineering of a target site-specific recombinase by a combined evolution- and structure-guided approach

Site-specific recombinases (SSRs) can perform DNA rearrangements, including deletions, inversions and translocations when their naive target sequences are placed strategically into the genome of an organism. Hence, in order to employ SSRs in heterologous hosts, their target sites have to be introduced into the genome of an organism before the enzyme can be practically employed. Engineered SSRs hold great promise for biotechnology and advanced biomedical applications, as they promise to extend the usefulness of SSRs to allow efficient and specific recombination of pre-existing, natural genomic sequences. However, the generation of enzymes with desired properties remains challenging. Here, we use substrate-linked directed evolution in combination with molecular modeling to rationally engineer an efficient and specific recombinase (sTre) that readily and specifically recombines a sequence present in the HIV-1 genome. We elucidate the role of key residues implicated in the molecular recognition mechanism and we present a rationale for sTre’s enhanced specificity. Combining evolutionary and rational approaches should help in accelerating the generation of enzymes with desired properties for use in biotechnology and biomedicine.

Next-generation LTR-specific Tre-recombinase targets a majority of HIV-1 isolates

HIV-1 integrates into the host chromosome and persists as a provirus flanked by long terminal repeats (LTR). To date, treatment regimens primarily target the virus enzymes, virus attachment or virus-cell fusion, but not the integrated provirus. Thus, current antiretroviral therapies (i.e. cART) cannot eradicate HIV-1, a fact that highlights the urgency of pursuing new strategies to find a cure for HIV/AIDS. Previously, we engineered an experimental LTR-specific recombinase (Tre-recombinase) that can effectively excise integrated HIV-1 proviral DNA from infected human cell cultures (Sarkar et al. 2007 Science 316:1912). Subsequently, we demonstrated highly significant antiviral activity of this HIV-1 subtype A-specific Tre in humanized mice (Hauber et al. 2013 PLOS pathogens 9:e1003587). Broad clinical application, however, requires availability of a tre-recombinase that recognizes a majority of clinical HIV-1 isolates.

Patient-adapted, specific activation of HIV-1 by customized TAL effectors (TALEs), a proof of principle study.

The major obstacle to cure infections with human immunodeficiency virus (HIV-1) is integrated proviral genomes, which are not eliminated by antiretroviral therapies (ART). Treatment approaches with latency-reversing agents (LRAs) aim at inducing provirus expression to tag latently-infected cells for clearance through viral cytopathic effects or cytotoxic T cell (CTL) responses. However, the currently tested LRAs reveal evident drawbacks as gene expression is globally induced and viral outgrowth is insecure. Here, we present transcription activator-like effector (TALE) proteins as potent tools to activate HIV-1 specifically. The large variety of circulating HIV-1 strains and, accordingly, integrated proviruses was addressed by the programmable DNA-specificity of TALEs. Using customized engineered TALEs, a substantial transcription activation and viral outgrowth was achieved with cells obtained from different HIV-1 patients. Our data suggest that TALEs may be useful tools in future strategies aimed at removing HIV-1 reservoirs.

Impact of ETIF Deletion on Safety and Immunogenicity of Equine Herpesvirus Type 1-Vectored Vaccines

ABSTRACT Heterologous gene transfer by viral vector systems is often limited by factors such as preexisting immunity, toxicity, low packaging capacity, or weak immunogenic potential. A novel viral vector system derived from equine herpesvirus type 1 (EHV-1) not only overcomes some of these obstacles but also promotes the robust expression of a delivered transgene and the induction of antigen-specific immune responses. Regarding an enhanced safety profile, we assessed the impact of the gene encoding the sole essential tegument protein, ETIF, on the replication and immunogenicity of recombinant EHVs. The deletion of ETIF severely attenuates replication in permissive RK13 cells and a human lung epithelial cell line but without influencing transgene expression. Whereas the intranasal administration of a recombinant luciferase EHV in BALB/c mice resulted in transgene expression in nasal cavities and lungs for 5 to 6 days, the ETIF deletion limited expression to 2 days and resulted in 30-fold-less luminescence. Attenuated replication was accompanied by a decreased capacity to induce CD8+ T cells against a delivered HIV Gag transgene in BALB/c mice following repeated intranasal application. However, a single subcutaneous immunization with a gag DNA vaccine primed specific T cells for substantial expansion by two subsequent intranasal booster immunizations with either the gag recombinant ETIF mutant or the parental virus. In addition to inducing Gag-specific serum antibodies, this prime-boost strategy clearly outperformed three sequential immunizations with the parental or EHV-ΔETIF virus or repeated DNA vaccination by inducing substantial specific secretory IgA (sIgA) titers.

Efficient transformation of primary human mesenchymal stromal cells by adenovirus early region 1 oncogenes

ABSTRACT Previous observations that human amniotic fluid cells (AFC) can be transformed by human adenovirus type 5 (HAdV-5) E1A/E1B oncogenes prompted us to identify the target cells in the AFC population that are susceptible to transformation. Our results demonstrate that one cell type corresponding to mesenchymal stem/stroma cells (hMSCs) can be reproducibly transformed by HAdV-5 E1A/E1B oncogenes as efficiently as primary rodent cultures. HAdV-5 E1-transformed hMSCs exhibit all properties commonly associated with a high grade of oncogenic transformation, including enhanced cell proliferation, anchorage-independent growth, increased growth rate, and high telomerase activity as well as numerical and structural chromosomal aberrations. These data confirm previous work showing that HAdV preferentially transforms cells of mesenchymal origin in rodents. More importantly, they demonstrate for the first time that human cells with stem cell characteristics can be completely transformed by HAdV oncogenes in tissue culture with high efficiency. Our findings strongly support the hypothesis that undifferentiated progenitor cells or cells with stem cell-like properties are highly susceptible targets for HAdV-mediated cell transformation and suggest that virus-associated tumors in humans may originate, at least in part, from infections of these cell types. We expect that primary hMSCs will replace the primary rodent cultures in HAdV viral transformation studies and are confident that these investigations will continue to uncover general principles of viral oncogenesis that can be extended to human DNA tumor viruses as well. IMPORTANCE It is generally believed that transformation of primary human cells with HAdV-5 E1 oncogenes is very inefficient. However, a few cell lines have been successfully transformed with HAdV-5 E1A and E1B, indicating that there is a certain cell type which is susceptible to HAdV-mediated transformation. Interestingly, all those cell lines have been derived from human embryonic tissue, albeit the exact cell type is not known yet. We show for the first time the successful transformation of primary human mesenchymal stromal cells (hMSCs) by HAdV-5 E1A and E1B. Further, we show upon HAdV-5 E1A and E1B expression that these primary progenitor cells exhibit features of tumor cells and can no longer be differentiated into the adipogenic, chondrogenic, or osteogenic lineage. Hence, primary hMSCs represent a robust and novel model system to elucidate the underlying molecular mechanisms of adenovirus-mediated transformation of multipotent human progenitor cells.