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HIV Cured In Mice? German Scientists Use Stem Cells To Cut Out Virus With 'Molecular Scissors'
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article excerpts & link follows below these press reports online:
Successful HIV treatment may be on the way - German researchers
Published time: December 19, 2013 13:34
http://rt.com
Biomedicine researchers at Dresden's Technical University have made several successful attempts to treat HIV virus with a new method which uses so-called ''molecular scissors'' to cut the virus from the DNA of infected cells.
The researchers have managed to manipulate the enzyme so that it can identify a particular sequence and remove it - and they say it is more than 90 percent effective in identifying the HIV virus this way, TheLocal.de reported.
"There are various methods and similar approaches, but removing the virus from infected cells is unique," said Professor Joachim Hauber, head of the antiviral strategy section at Dresden Technical University's partner research lab at Hamburg's Heinrich Pette Institute, told the German daily.
Molecular scissors that carry out the cutting could be ready to use in 10 years as a somatic genetic therapy (when a patient's cells are altered and put back into the body).
The theory is that the genetically altered immune cells would reproduce, cut the HIV out of infected cells, and as a result, enable them to function again.
The Dresden researchers have put the theory into practice, as the method works - at least on mice.
"The amount of virus was clearly reduced, and was even no longer to be found in the blood," Hauber said.
An enzyme created especially via mutation and selection identifies HIV.
"It is one of the most exciting things of all," he said. "There is a vague hope of cure, but that must first be proven."
It can be established if the newly-discovered method would function with people only in clinical trials, the money for which haven't been found.
Another issue related to the HIV drug is that pharmaceutical companies have shown little interest in it.
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http://uk.news.yahoo.com
President of the German Aids Society, Professor Jurgen Rockstroh said he hoped funding could be found for further work on the approach.
"It is one of the most exciting things of all," he said. "There is a vague hope of cure, but that must first be proven."
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HIV Cured In Mice? German Scientists Use Stem Cells To Cut Out Virus With 'Molecular Scissors'
By Chris Weller | Dec 19, 2013 11:44 AM EDT
http://www.medicaldaily.com
Scientists from Germany's Dresden University of Technology have discovered a potential treatment method that not only delayed the growth of HIV cells in laboratory mice, but fully cured some of the subjects, according to a new study published in PLoS Pathogens.
The process utilizes, what researchers call, "molecular scissors." These scissors (essentially an HIV-cutting enzyme, known as tre-recombinase) help weed out the virus from infected cells after scientists have injected it into the host with a combination of genetically altered stem cells. This process is known as somatic genetic therapy, and it's one the research team claims could break new ground in the search for a robust, effective cure for one of the world's deadliest viruses.
"There are various methods and similar approaches, but removing the virus from infected cells is unique," Professor Joachim Hauber, head of the antiviral strategy section at Hamburg's Heinrich Pette Institute, told The Local, adding that the method is unique because it is only one so far to reverse an HIV infection yet still leave the treated cells healthy.
The entire process begins with extracting blood samples from the humanized mice - i.e., mice that have been introduced to human genes, cells, tissues, or organs. These samples are then harvested for their stems cells, which can then be used to form new, genetically altered blood cells. When scientists reintroduce the newly engineered samples, which now contain the HIV-cutting enzyme, the enzyme locates the genes on the end of the virus' genome, removes it from the host DNA's genome, and effectively cures the cell of infection.
According to Hauber, the proposed theory sees the genetically altered immune cells reproducing, so that all future cases of HIV would be wiped clean from the body through the use of these "scissors." "In the humanized mice model, he said, "the amount of virus was clearly reduced, and even no longer to be found in the blood."
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"It is one of the most exciting things of all," he added. "There is a vague hope of cure, but that must first be proven." Use of molecular scissors in clinical trials may still be a decade away, the team notes.
Despite the advances made by the current study, the research team expressed little hope that their findings would advance in the current climate. Large prescription drug companies have a vested interest in keeping patients on their medications - repeat customers, in essence - and any breakthrough options that hint at the "C word" are actually a negative, even with the incomparable benefits of worldwide cure.
"The potential is not being used," Hauber said, referring to the large banks of scientific knowledge concerning HIV/AIDS. According to the World Health Organization (WHO), the disease has already claimed 36 million lives so far, and another 35 million were living with the disease in 2012. More harrowing even is that the majority of disease carriers live in sub-Saharan Africa, a region of the world with scant access to proper treatment options. Nearly one in 20 people live with HIV in this region, which, in itself, makes up 69 percent of the world's infected population.
In the U.S. alone, an estimated 1,148,200 people 13 years and older were living with an HIV infection. Of those, 18 percent (or 207,600) hadn't yet been diagnosed. That equates to a prevalence in sub-Saharan Africa roughly 17 times greater than in the U.S.
Source: Hauber I, Hofmann-Sieber H, Chemnitz J. Highly Significant Antiviral Activity of HIV-1 LTR-Specific Tre-Recombinase in Humanized Mice. PLoS Pathogens. 2013
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PLoS Pathogens, link to article:
Highly Significant Antiviral Activity of HIV-1 LTR-Specific Tre-Recombinase in Humanized Mice
http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003587
"Clearly, it is not expected that HIV-1 can be eradicated by Tre activity alone. As outlined above, future HIV eradication strategies are conceived to be a combination of various antiviral approaches (e.g. drug-based and gene therapies), host immunity enhancing treatments (e.g. therapeutic vaccination approaches), and purging attempts to overcome latency [51]-[53]. In summary, our data support the notion that Tre-recombinase technology can be a valuable component of such a multi-tiered strategy to treat HIV-infected patients."
"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.
......the most direct approach to eliminating virus reservoirs is to remove HIV genomes from infected cells. As previous studies suggested, this may be achievable by Tre-recombinase, an engineered enzyme that can excise integrated HIV from host cell chromosomes. The present work analyzes the expression of Tre-recombinase in human cells and demonstrates highly accurate Tre activity in complete absence of Tre-related cytopathic effects. Furthermore, in vivo analysis of Tre-recombinase demonstrates highly significant antiviral effects of Tre in HIV-infected humanized mice. The presented data suggest that Tre-recombinase might become a valuable component of a future therapy that aims at virus eradication."
It is generally believed that the main hurdle to virus eradication is the persisting HIV-1 infection in latent reservoirs, particularly in memory CD4+ T cells (reviewed in [9]-[15]). Latently HIV-1-infected resting CD4+ T cells are apparently established early in infection. One current strategy to eliminate this pool of long-lived cells aims to specifically activate the transcriptionally quiescent provirus (i.e. the integrated replication-competent HIV-1 genome), for example by modifying its chromatin structure through histone deacetylase (HDAC) inhibitors (reviewed in [12], [13], [15]-[17]). Upon HDAC inhibitor-induced HIV-1 antigen expression, it is expected that these cells either experience HIV-1-induced cell death or are eliminated by cytotoxic T cells (CTLs). It is fair to assume that such purging strategies would greatly benefit from a technology that can concurrently remove integrated HIV-1 from the pool of productively infected cells, thereby restoring, or at least improving the patient's immune function.
A novel strategy to remove integrated HIV-1 is based on a tailored site-specific recombinase (Tre), derived by molecular evolution of the bacteriophage recombinase Cre [18]-[20]. Tre targets a specific 34 bp sequence (loxLTR) derived from a primary HIV-1 strain [21] located in the proviral LTR regions, resulting in excision of the integrated proviral DNA from the genome of infected cultured cells [18]. This process not only suppresses viral replication, but in theory may also help eradicate HIV from an infected individual (reviewed in [22]).
Administering Tre-recombinase to patients will most likely require a gene therapy approach. In principle, genetic therapies against HIV either modify the patient's peripheral CD4+ T cells or patient-derived CD34+ hematopoietic stem cells (HSC) [23]-[25]. It is anticipated that the former strategy would lead to beneficial antiviral, although transient effects. The latter application will presumably be the preferred strategy in Tre-based virus eradication approaches, since, in theory, it allows perpetual repopulation of the patient's hematopoietic system with Tre-expressing HIV-1 target cells. These cells may be selected in vivo [26], since upon de novo infection they are able to remove the integrated HIV-1 proviral DNA, and thus remain functionally immune competent.
Independently of the selected gene therapy strategy, and prior to its potential use in HIV-infected patients, vector technology has to be developed that allows safe and efficient gene transfer followed by reliable transgene expression in target cells. Moreover, the absence of cytopathic and/or genotoxic effects upon vector-mediated Tre expression, and the accurate excision of HIV proviral DNA from chromosomal integration sites has to be demonstrated. Finally, the antiviral effects of Tre-recombinase have to be shown in vivo, i.e. in an appropriate animal model for HIV-1 infection. All these analyses will be of utmost importance for developing a potential Tre-based therapy to treat HIV infection.
Discussion
The clinical development of HAART has been one of the great successes in modern medicine. However, the fact that HAART cannot eradicate HIV [7], [8] makes investigating novel antiviral strategies a prerequisite for developing a future cure for HIV infection [7], [9], [12]. In effect, gene therapy strategies represent a technology holding high promise for future antiviral disease treatments [22]-[25]. Indeed, various RNA-based technologies are currently being investigated in vivo, including, for example, the expression of RNA aptamers, siRNAs and shRNAs, TAR decoys, and ribozymes [42]-[44]. Moreover, the expression of membrane-bound fusion inhibitors is another appealing antiviral strategy [45], [46]. These approaches efficiently suppress virus replication, and thus reduce viral loads for extended periods of time. Another promising strategy appears to be disruption of the CCR5 gene [47], [48], for example by expressing engineered zinc finger nucleases (ZFN) [20]. In humanized mice transplanted with either CD4+ T cells or CD34+ HSC, ZFN-mediated CCR5 disruption has been shown to confer resistance to de novo infection by CCR5-tropic HIV-1, thereby controlling virus replication [49], [50].
In contrast, an antiviral strategy based on Tre-recombinase is independent of virus coreceptor usage (i.e. tropism) and can target cells that are already infected with HIV [18], [34]. Importantly, Tre-mediated provirus excision allows reversal of HIV infection at the cellular level, thereby avoiding viral cytopathic effects (e.g. effects associated with viral antigen expression) and possibly restoring host cell function. As shown here, Tre expression mediated highly significant antiviral effects, which were equally observed in animals engrafted with Tre-expressing CD4+ T cells or Tre-expressing CD34+ HSC. With respect to future clinical studies, this Tre effect is particularly impressive, since the animals had been transplanted with unselected cell pools, where only a fraction of the T cells (~60%) or the HSC (~30%) harbored the Tre expressing lentiviral vector. Apparently, Tre-mediated protection of only a subpopulation of HIV-1 target cells suffices to achieve significant antiviral effects in vivo. This may be explained by the in vivo selection of gene vector-transduced cells as well as potential bystander effects [22]-[25].
The results presented here suggest that Tre vectors are promising antiviral reagents for therapies based on the genetic modification of both peripheral T cells and hematopoietic stem cells. Clearly, the procedure for ex vivo treatment of peripheral T cells is comparably less complicated, and aims at a functional cure by achieving long-term control of HIV, preferably in the absence of HAART [7]. In contrast, the development of a sterilizing cure that eradicates HIV-1 from an infected organism, if achievable at all, most likely requires a highly complex strategy, involving the autologous transplantation of gene-modified HSC [22], [25]. It is then expected that the elimination of all HIV-infected cells may eventually depend on reconstituting the patient's immune functions, a process that presumably requires additional and potentially gene therapy-unrelated approaches such as, for example, immune activation and/or purging strategies [51]-[54]. It is likely that such multi-pronged eradication approaches will benefit from Tre-mediated provirus excision in the patient's immune effector cells (e.g. CD4+ T cells).
Important safety issues related to gene therapies are generally connected with potential cytopathic effects caused by the respective transgene and/or the vector technology used. The latter was addressed here by using an advanced lentiviral SIN vector design where transgene expression was placed under the control of a Tat-inducible promoter, limiting its expression to HIV-infected cells. This strategy circumvents a major shortcoming of various antiviral gene therapies, the continued expression of foreign transgenes [23], thereby minimizing undesired transgene-related side effects such as immunogenicity.
Obviously, expression of Tre-recombinase from a Tat-inducible promoter presumably precludes provirus excision in latently infected cells. It is therefore conceived that in clinical virus eradication approaches Tat-responsive Tre-expressing vectors will only be used in combination with purging drugs that, as previously shown, not only specifically activate the transcription of otherwise quiescent proviral genomes [55], [56], but, at the same time, will also enable Tat-mediated Tre expression from the vector used in the present study. In this context it is also important to note that a recent study demonstrated that drug-induced purging alone does not result in the elimination of patient-derived infected resting CD4+ T cells, even when autologous CTLs were present [57]. In fact, after virus reactivation these cells where only killed when the HIV-specific CTLs were pre-stimulated, suggesting that virus eradication depends at least on a combination of purging drugs with therapeutic vaccination strategies [57]. It is expected that such an approach would further benefit from the inclusion of an additional anti-HIV gene therapy [25], [58], such as Tre-mediated provirus excision [22]. It is also noted that recent computational modeling of HIV dynamics in the presence of a replication incompetent Tre-recombinase-expressing therapeutic vector suggested that such an approach may indeed clear all HIV from the system in the long term [59].
Clearly, our study does not investigate the efficacy of Tre for latent proviruses. This is a significant limitation that will be addressed in the future. Particularly, it will be of interest to see whether a residual Tat level exists in latently infected cells that enables Tre expression by the current vector design. Alternatively, Tat-independent vectors that employ drug-inducible promoters may permit conditional Tre expression in resting cells. For example, advanced doxycycline-responsive promoter systems hold the promise to further increase biosafety of gene therapies by actively controlling transgene expression [60]. In this context one may also conceive the direct delivery of Tre-recombinase into patients, for example by applying Tre-containing virus like particles [61]. It is noted, that excision of proviral DNA by recombinant cell permeable Tre-recombinase has been already demonstrated in cell cultures [34]. Thus, such advanced Tre delivery systems are conceived to play an important role in the future, particularly for targeting latently infected resting cells.
Another safety aspect that should not be underestimated is based on Tre's pronounced target site specificity. The fact that the site-specific recombination process mediated by such Cre-derived enzymes neither produces free DNA ends (e.g. double-strand breaks) nor requires additional host factors [62], minimizes the oncogenic risk. In agreement, the advanced molecular cytogenetic analyses presented here demonstrate the absence of Tre-related genome-wide off-target effects. This distinguishes Tre-recombinase from CCR5-specific ZFNs, which may suffer from off-target cleavage specificities [49], [63], [64].
Nevertheless, virus entry inhibition by CCR5 knockout represents a highly attractive antiviral strategy that may be exploited to its full extent when combined with Tre-recombinase technology, thereby not only blocking de novo infection but also targeting already infected cells for provirus excision.
The presented data suggest that antiviral gene therapies are feasible using conditionally expressed, engineered Tre-recombinases that precisely remove HIV-1 proviral DNA without cytopathic effects. Antiviral in vivo activity was observed by transduction of both CD4+ T cells and CD34+ HSC. Particularly the latter stem cell-based approach may be a valuable component of future eradication strategies to cure HIV [22], [25]. The fact that the current Tre-recombinase recognizes particularly HIV-1 subtype A isolates may limit its broad application. However, the recent identification of highly conserved HIV-1 LTR sequences [65] in combination with a novel loxLTR search tool [35] now permits the engineering of advanced Tre-recombinases with activity against the majority of HIV-1 variants.
Clearly, it is not expected that HIV-1 can be eradicated by Tre activity alone. As outlined above, future HIV eradication strategies are conceived to be a combination of various antiviral approaches (e.g. drug-based and gene therapies), host immunity enhancing treatments (e.g. therapeutic vaccination approaches), and purging attempts to overcome latency [51]-[53]. In summary, our data support the notion that Tre-recombinase technology can be a valuable component of such a multi-tiered strategy to treat HIV-infected patients.
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