Robert A. Wu

Foamy combinatorial anti-HIV vectors with MGMTP140K potently inhibit HIV-1 and SHIV replication and mediate selection in vivo

Highly active antiretroviral therapy has greatly reduced the morbidity and mortality from human immunodeficiency virus (HIV) infection, but AIDS continues to be a serious health problem worldwide. Despite enormous efforts to develop a vaccine, there is still no cure, and alternative approaches including gene therapy should be explored. In this study we developed and compared combinatorial foamy virus (FV) anti-HIV vectors that also express a mutant methylguanine methyltransferase (MGMTP140K) transgene to increase the percentage of gene-modified cells after transplantation. These FV vectors inhibit replication of HIV-1 and also the simian immunodeficiency virus/HIV-1 (SHIV) chimera that can be used in monkey AIDS gene therapy studies. We identified a combinatorial FV vector that expresses 3 anti-HIV transgenes and inhibits viral replication by over 4 logs in a viral challenge assay. This FV anti-HIV vector expresses an HIV fusion inhibitor and two short hairpin RNAs (shRNAs) targeted to HIV-1 tat and rev, and can be produced at high titer (3.8 × 107 transducing units ml−1) using improved helper plasmids suitable for clinical use. Using a competitive repopulation assay, we show that human CD34+ cells transduced with this combinatorial FV vector efficiently engraft in a mouse xenotransplantation model, and that the percentage of transduced repopulating cells can be increased after transplantation.

Protection of Stem Cell-Derived Lymphocytes in a Primate AIDS Gene Therapy Model after In Vivo Selection

Background There is currently no effective AIDS vaccine, emphasizing the importance of developing alternative therapies. Recently, a patient was successfully transplanted with allogeneic, naturally resistant CCR5-negative (CCR5Δ32) cells, setting the stage for transplantation of naturally resistant, or genetically modified stem cells as a viable therapy for AIDS. Hematopoietic stem cell (HSC) gene therapy using vectors that express various anti-HIV transgenes has also been attempted in clinical trials, but inefficient gene transfer in these studies has severely limited the potential of this approach. Here we evaluated HSC gene transfer of an anti-HIV vector in the pigtailed macaque (Macaca nemestrina) model, which closely models human transplantation. Methods and Findings We used lentiviral vectors that inhibited both HIV-1 and simian immunodeficiency virus (SIV)/HIV-1 (SHIV) chimera virus infection, and also expressed a P140K mutant methylguanine methyltransferase (MGMT) transgene to select gene-modified cells by adding chemotherapy drugs. Following transplantation and MGMT-mediated selection we demonstrated transgene expression in over 7% of stem-cell derived lymphocytes. The high marking levels allowed us to demonstrate protection from SHIV in lymphocytes derived from gene-modified macaque long-term repopulating cells that expressed an HIV-1 fusion inhibitor. We observed a statistically significant 4-fold increase of gene-modified cells after challenge of lymphocytes from one macaque that received stem cells transduced with an anti-HIV vector (p<0.02, Students t-test), but not in lymphocytes from a macaque that received a control vector. We also established a competitive repopulation assay in a second macaque for preclinical testing of promising anti-HIV vectors. The vectors we used were HIV-based and thus efficiently transduce human cells, and the transgenes we used target HIV-1 genes that are also in SHIV, so our findings can be rapidly translated to the clinic. Conclusions Here we demonstrate the ability to select protected HSC-derived lymphocytes in vivo in a clinically relevant nonhuman primate model of HIV/SHIV infection. This approach can now be evaluated in human clinical trials in AIDS lymphoma patients. In this patient setting, chemotherapy would not only kill malignant cells, but would also increase the number of MGMTP140K-expressing HIV-resistant cells. This approach should allow for high levels of HIV-protected cells in AIDS patients to evaluate AIDS gene therapy.

Human telomerase specialization for repeat synthesis by unique handling of primer‐template duplex

With eukaryotic genome replication, incomplete telomere synthesis results in chromosome shortening and eventual compromise of genome stability. Telomerase counteracts this terminal sequence loss by synthesizing telomeric repeats through repeated cycles of reverse transcription of its internal RNA template. Using human telomerase domain‐complementation assays for telomerase reverse transcriptase protein (TERT) and RNA in combination with the first direct footprinting assay for telomerase association with bound DNA, we resolve mechanisms by which TERT domains and RNA motifs direct repeat synthesis. Surprisingly, we find that product‐template hybrid is sensed in a length‐ and sequence‐dependent manner to set the template 5′ boundary. We demonstrate that the TERT N‐terminal (TEN) domain determines active‐site use of the atypically short primer‐template hybrid necessary for telomeric‐repeat synthesis. Also against expectation, we show that the remainder of TERT (the TERT ring) supports functional recognition and physical protection of single‐stranded DNA adjacent to the template hybrid. These findings establish unprecedented polymerase recognition specificities for DNA‐RNA hybrid and single‐stranded DNA and suggest a new perspective on the mechanisms of telomerase specialization for telomeric‐repeat synthesis.

Cocal-pseudotyped Lentiviral Vectors Resist Inactivation by Human Serum and Efficiently Transduce Primate Hematopoietic Repopulating Cells

Lentiviral vectors are established as efficient and convenient vehicles for gene transfer. They are almost always pseudotyped with the envelope glycoprotein of vesicular stomatitis virus (VSV-G) due to the high titers that can be achieved, their stability, and broad tropism. We generated a novel cocal vesiculovirus envelope glycoprotein plasmid and compared the properties of lentiviral vectors pseudotyped with cocal, VSV-G, and a modified feline endogenous retrovirus envelope glycoprotein (RD114/TR). Cocal-pseudotyped lentiviral vectors can be produced at titers as high as with VSV-G, have a broad tropism, and are stable, allowing for efficient concentration by centrifugation. Additionally, cocal vectors are more resistant to inactivation by human serum than VSV-G-pseudotyped vectors, and efficiently transduce human CD34(+) nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse-repopulating cells (SRCs), and long-term primate hematopoietic repopulating cells. These studies establish the potential of cocal-pseudotyped lentiviral vectors for a variety of scientific and therapeutic gene transfer applications, including in vivo gene delivery and hematopoietic stem cell (HSC) gene therapy.

Stem Cell Selection In Vivo Using Foamy Vectors Cures Canine Pyruvate Kinase Deficiency

Background Hematopoietic stem cell (HSC) gene therapy has cured immunodeficiencies including X-linked severe combined immunodeficiency (SCID-X1) and adenine deaminase deficiency (ADA). For these immunodeficiencies corrected cells have a selective advantage in vivo, and low numbers of gene-modified cells are sufficient to provide therapeutic benefit. Strategies to efficiently transduce and/or expand long-term repopulating cells in vivo are needed for treatment of diseases that require higher levels of corrected cells, such as hemoglobinopathies. Here we expanded corrected stem cells in vivo in a canine model of a severe erythroid disease, pyruvate kinase deficiency. Methodology/Principal Findings We used a foamy virus (FV) vector expressing the P140K mutant of methylguanine methyltransferase (MGMTP140K) for in vivo expansion of corrected hematopoietic repopulating cells. FV vectors are attractive gene transfer vectors for hematopoietic stem cell gene therapy since they efficiently transduce repopulating cells and may be safer than more commonly used gammaretroviral vectors. Following transplantation with HSCs transduced ex vivo using a tri-cistronic FV vector that expressed EGFP, R-type pyruvate kinase, and MGMTP140K, we were able to increase marking from approximately 3.5% to 33% in myeloid long-term repopulating cells resulting in a functional cure. Conclusions/Significance Here we describe in one affected dog a functional cure for a severe erythroid disease using stem cell selection in vivo. In addition to providing a potential cure for patients with pyruvate kinase deficiency, in vivo selection using foamy vectors with MGMTP140K has broad potential for several hematopoietic diseases including hemoglobinopathies.

Single-molecule imaging of telomerase reverse transcriptase in human telomerase holoenzyme and minimal RNP complexes

Telomerase synthesizes chromosome-capping telomeric repeats using an active site in telomerase reverse transcriptase (TERT) and an integral RNA subunit template. The fundamental question of whether human telomerase catalytic activity requires cooperation across two TERT subunits remains under debate. In this study, we describe new approaches of subunit labeling for single-molecule imaging, applied to determine the TERT content of complexes assembled in cells or cell extract. Surprisingly, telomerase reconstitutions yielded heterogeneous DNA-bound TERT monomer and dimer complexes in relative amounts that varied with assembly and purification method. Among the complexes, cellular holoenzyme and minimal recombinant enzyme monomeric for TERT had catalytic activity. Dimerization was suppressed by removing a TERT domain linker with atypical sequence bias, which did not inhibit cellular or minimal enzyme assembly or activity. Overall, this work defines human telomerase DNA binding and synthesis properties at single-molecule level and establishes conserved telomerase subunit architecture from single-celled organisms to humans. DOI: http://dx.doi.org/10.7554/eLife.08363.001

Sequence specificity of human telomerase

DNA replication machinery copies an entire genome with remarkable speed and accuracy in preparation for each cell division. However, DNA polymerase unidirectionality and requirement for a free 3′ hydroxyl group render these enzymes incapable of duplicating the extreme ends of eukaryotic linear chromosomes. This deficiency leads to a gradual loss of terminal sequence with each round of replication that threatens the integrity of essential genetic information. This calamity is avoided through the function of telomeres, typically structures of short repeating sequence added to chromosome 3′ ends by telomerase (1). Soon after its discovery as a polymerase, telomerase was found to reverse transcribe a template sequence contained within the telomerase integral RNA subunit TER (2). Telomerase biochemical activity has proven to be unique even among reverse transcriptases (RTs) in features including the release of single-stranded rather than duplex product DNA, which is necessary for regenerating the template for telomerase’s distinctive repeat synthesis processivity (3). How telomerase gained new types of nucleic acid handling specificity relative to other RTs has been the subject of intense research, much of which has focused on the structure and function of nontemplate specializations of TER and unique domains of the telomerase protein RT subunit TERT (4). In PNAS, Brown et al. present surprising evidence of a previously missed specialization of human telomerase (5). Using elegant and incisive biochemical assays, the authors demonstrate that this enzyme reads sequence cues within the product–template duplex to dramatically influence the specificity of product synthesis. The work reveals an unanticipated divergence of telomerase from other polymerases in active site adaptation for its cellular duty. The diverse template and substrate maneuvers that occur during repeat synthesis constitute a telomerase catalytic cycle (Fig. 1 A ). A primer 3′ end base pairs at one end of the TER template … [↵][1]1To whom correspondence may be addressed. Email: rawu{at}berkeley.edu or kcollins{at}berkeley.edu. [1]: #xref-corresp-1-1

DNA-binding determinants and cellular thresholds for human telomerase repeat addition processivity

The reverse transcriptase telomerase adds telomeric repeats to chromosome ends. Purified human telomerase catalyzes processive repeat synthesis, which could restore the full ~100 nucleotides of (T2AG3)n lost from replicated chromosome ends as a single elongation event. Processivity inhibition is proposed to be a basis of human disease, but the impacts of different levels of processivity on telomere maintenance have not been examined. Here, we delineate side chains in the telomerase active‐site cavity important for repeat addition processivity, determine how they contribute to duplex and single‐stranded DNA handling, and test the cellular consequences of partial or complete loss of repeat addition processivity for telomere maintenance. Biochemical findings oblige a new model for DNA and RNA handling dynamics in processive repeat synthesis. Biological analyses implicate repeat addition processivity as essential for telomerase function. However, telomeres can be maintained by telomerases with lower than wild‐type processivity. Furthermore, telomerases with low processivity dramatically elongate telomeres when overexpressed. These studies reveal distinct consequences of changes in telomerase repeat addition processivity and expression level on telomere elongation and length maintenance.

Cryo-EM structure of substrate-bound human telomerase holoenzyme.

The enzyme telomerase adds telomeric repeats to chromosome ends to balance the loss of telomeres during genome replication. Telomerase regulation has been implicated in cancer, other human diseases, and ageing, but progress towards clinical manipulation of telomerase has been hampered by the lack of structural data. Here we present the cryo-electron microscopy structure of the substrate-bound human telomerase holoenzyme at subnanometre resolution, showing two flexibly RNA-tethered lobes: the catalytic core with telomerase reverse transcriptase (TERT) and conserved motifs of telomerase RNA (hTR), and an H/ACA ribonucleoprotein (RNP). In the catalytic core, RNA encircles TERT, adopting a well-ordered tertiary structure with surprisingly limited protein–RNA interactions. The H/ACA RNP lobe comprises two sets of heterotetrameric H/ACA proteins and one Cajal body protein, TCAB1, representing a pioneering structure of a large eukaryotic family of ribosome and spliceosome biogenesis factors. Our findings provide a structural framework for understanding human telomerase disease mutations and represent an important step towards telomerase-related clinical therapeutics.A cryo-electron microscopy structure of the substrate-bound human telomerase holoenzyme, which lengthens the protective caps on chromosomes.

Endogenous Telomerase Reverse Transcriptase N-Terminal Tagging Affects Human Telomerase Function at Telomeres In Vivo

ABSTRACT Telomerase action at telomeres is essential for the immortal phenotype of stem cells and the aberrant proliferative potential of cancer cells. Insufficient telomere maintenance can cause stem cell and tissue failure syndromes, while increased telomerase levels are associated with tumorigenesis. Both pathologies can arise from only small perturbation of telomerase function. To analyze telomerase at its low endogenous expression level, we genetically engineered human pluripotent stem cells (hPSCs) to express various N-terminal fusion proteins of the telomerase reverse transcriptase from its endogenous locus. Using this approach, we found that these modifications can perturb telomerase function in hPSCs and cancer cells, resulting in telomere length defects. Biochemical analysis suggests that this defect is multileveled, including changes in expression and activity. These findings highlight the unknown complexity of telomerase structural requirements for expression and function in vivo.