Dirk Lindemann

A new approach to transcription factor screening for reprogramming of fibroblasts to cardiomyocyte-like cells

The simultaneous overexpression of several transcription factors has emerged as a successful strategy to convert fibroblasts into other cell types including pluripotent cells, neurons, and cardiomyocytes. The selection and screening of factors are critical, and have often involved testing a large pool of transcription factors, followed by successive removal of single factors. Here, to identify a cardiac transcription factor combination facilitating mouse fibroblast reprogramming into cardiomyocytes, we directly screened all triplet combinations of 10 candidate factors combined with a Q-PCR assay reporting induction of multiple cardiac-specific genes. Through this screening method the combination of Tbx5, Mef2c, and Myocd was identified to upregulate a broader spectrum of cardiac genes compared to the combination of Tbx5, Mef2c, and Gata4 that was recently shown to induce reprogramming of fibroblasts into cardiomyocytes. Cells cotransduced with Tbx5, Mef2c, Myocd expressed cardiac contractile proteins, had cardiac-like potassium and sodium currents and action potentials could be elicited. In summary the alternative screening approach that is presented here avoided the elimination of transcription factors whose potency is masked in complex transcription factor mixes. Furthermore, our results point to the importance of verifying multiple lineage specific genes when assessing reprogramming.

Comparison of three retroviral vector systems for transduction of nonobese diabetic/severe combined immunodeficiency mice repopulating human CD34+ cord blood cells

The use of recombinant vectors based on wild-type viruses that are absent in humans and are not associated with any disease in their natural animal hosts or in accidentally infected humans would add an additional level of safety for human somatic gene therapy approaches. These criteria are fulfilled by foamy viruses (FVs), a family of complex retroviruses whose members are widely found among mammals and are apathogenic in all hosts. Here, we show by comparison of identically designed vector constructs that recombinant retroviral vectors based on FVs were as efficient as lentiviral vectors in transducing nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice repopulating human CD34(+) cord blood (CB) cells. The FV vector was able to achieve gene transfer levels up to 84% of engrafted human cells in a short overnight transduction protocol. In contrast, without prestimulation of the target cells, a human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector pseudotyped with gibbon ape leukemia virus envelope (GALV Env) was nearly as inefficient as murine leukemia virus (MLV)-based oncoretroviral vectors in transducing NOD/SCID repopulating cells. The same HIV vector pseudotyped with the vesicular stomatitis virus glycoprotein G (VSV-G) achieved high marking efficiency. Clonality analysis of bone marrow samples showed oligoclonal hematopoiesis with single to multiple insertions per cell, both for FV and HIV vectors. These data demonstrate that vectors based on FVs warrant further investigation and development for medical use.

Improved Primate Foamy Virus Vectors and Packaging Constructs

ABSTRACT Foamy virus (FV) vectors that have minimal cis-acting sequences and are devoid of residual viral gene expression were constructed and analyzed by using a packaging system based on transient cotransfection of vector and different packaging plasmids. Previous studies indicated (i) that FV gag gene expression requires the presence of the R region of the long terminal repeat and (ii) that RNA from packaging constructs is efficiently incorporated into vector particles. Mutants with changes in major 5′ splice donor (SD) site located in the R region identified this sequence element as responsible for regulating gag gene expression by an unidentified mechanism. Replacement of the FV 5′ SD with heterologous splice sites enabled expression of the gag and pol genes. The incorporation of nonvector RNA into vector particles could be reduced to barely detectable levels with constructs in which the human immunodeficiency virus 5′ SD or an unrelated intron sequence was substituted for the FV 5′ untranslated region and in which gag expression and pol expression were separated on two different plasmids. By this strategy, efficient vector transfer was achieved with constructs that have minimal genetic overlap.

A Particle-Associated Glycoprotein Signal Peptide Essential for Virus Maturation and Infectivity

ABSTRACT Signal peptides (SP) are key determinants for targeting glycoproteins to the secretory pathway. Here we describe the involvement in particle maturation as an additional function of a viral glycoprotein SP. The SP of foamy virus (FV) envelope glycoprotein is predicted to be unusually long. Using an SP-specific antiserum, we demonstrate that its proteolytic removal occurs posttranslationally by a cellular protease and that the major N-terminal cleavage product, gp18, is found in purified viral particles. Analysis of mutants in proposed signal peptidase cleavage positions and N-glycosylation sites revealed an SP about 148 amino acids (aa) in length. FV particle release from infected cells requires the presence of cognate envelope protein and cleavage of its SP sequence. An N-terminal 15-aa SP domain with two conserved tryptophan residues was found to be essential for the egress of FV particles. While the SP N terminus was found to mediate the specificity of FV Env to interact with FV capsids, it was dispensable for Env targeting to the secretory pathway and FV envelope-mediated infectivity of murine leukemia virus pseudotypes.

Extracellular signal regulated kinase 5 (ERK5) is required for the differentiation of muscle cells

Extracellular signal regulated kinase 5 (ERK5) is a novel member of the mitogen‐activated protein kinase (MAPK) family with a poorly defined physiological function. Since ERK5 and its upstream activator MEK5 are abundant in skeletal muscle we examined a function of the cascade during muscle differentiation. We show that ERK5 is activated upon induction of differentiation in mouse myoblasts and that selective activation of the pathway results in promoter activation of differentiation‐specific genes. Moreover, myogenic differentiation is completely blocked when ERK5 expression is inhibited by antisense RNA. Thus, we conclude that the MEK5/ERK5 MAP kinase cascade is critical for early steps of muscle cell differentiation.

A1 expression is stimulated by CD40 in B cells and rescues WEHI 231 cells from anti-IgM-induced cell death

Engagement of the antigen receptor on murine immature B cells leads to growth arrest followed by apoptosis. Concomitant signaling through CD40 sustains proliferation and rescues the cells from apoptosis. We show here that cross‐linking CD40 stimulates the expression of A1, a member of the anti‐apoptotic Bcl‐2 family, in primary murine B lymphocytes. CD40‐dependent stimulation of A1 was confirmed in WEHI 231 cells, an immature murine B cell lymphoma line. We transduced WEHI 231 cells with a bicistronic recombinant retroviral vector coding for A1 and a chimeric selection marker comprising the enhanced yellow fluorescent protein and the zeocin resistance protein. A1‐transduced WEHI 231 cells showed a significant higher survival rate after engagement of the antigen receptor. In contrast, constitutive expression of A1 did not abrogate anti‐IgM‐induced c‐myc down‐regulation. Consistant with this, A1 did not release anti‐IgM‐induced cell cycle arrest. Our data indicate that CD40‐stimulated A1 expression permits WEHI 231 cells to survive in the presence of anti‐IgM antibodies and suggests a protective role for A1 in antigen receptor‐mediated apoptosis in B cells.

Type III TGF-β receptor-independent signalling of TGF-β2 via TβRII-B, an alternatively spliced TGF-β type II receptor

Transforming growth factor‐β (TGF‐β) signals through membrane‐bound serine/threonine kinase receptors, which upon stimulation phosphorylate Smad proteins and thereby trigger their nuclear translocation and transcriptional activity. Although the three mammalian isoforms of TGF‐β are highly homologous at the level of sequence, analysis of their in vivo function by gene knockouts revealed striking differences, suggesting no significant functional redundancy between TGF‐β1, ‐2 and ‐3. While signal transduction by TGF‐β1 has been well characterized, receptor binding and activation by the TGF‐β2 isoform is less well understood. Here, we show that TβRII‐B, an alternatively spliced variant of the TGF‐β type II receptor, is a TGF‐β2 binding receptor, which mediates signalling via the Smad pathway in the absence of any TGF‐β type III receptor (TβRIII). L6 cells lacking endogenous TβRIII as well as TβRII‐B do not respond to TGF‐β2. Transfection of these cells with TβRII‐B restores TGF‐β2 sensitivity. The expression of TβRII‐B is restricted to cells originating from tissues such as bone where the isoform TGF‐β2 has a predominant role. This reflects the importance of this receptor in TGF‐β isoform‐specific signalling.

Mouse SAMHD1 Has Antiretroviral Activity and Suppresses a Spontaneous Cell-Intrinsic Antiviral Response

SUMMARY Aicardi-Goutières syndrome (AGS), a hereditary autoimmune disease, clinically and biochemically overlaps with systemic lupus erythematosus (SLE) and, like SLE, is characterized by spontaneous type I interferon (IFN) production. The finding that defects of intracellular nucleases cause AGS led to the concept that intracellular accumulation of nucleic acids triggers inappropriate production of type I IFN and autoimmunity. AGS can also be caused by defects of SAMHD1, a 3′ exonuclease and deoxy-nucleotide (dNTP) triphosphohydrolase. Human SAMHD1 is an HIV-1 restriction factor that hydrolyzes dNTPs and decreases their concentration below the levels required for retroviral reverse transcription. We show in gene-targeted mice that also mouse SAMHD1 reduces cellular dNTP concentrations and restricts retroviral replication in lymphocytes, macrophages, and dendritic cells. Importantly, the absence of SAMHD1 triggered IFN-β-dependent transcriptional upregulation of type I IFN-inducible genes in various cell types indicative of spontaneous IFN production. SAMHD1-deficient mice may be instrumental for elucidating the mechanisms that trigger pathogenic type I IFN responses in AGS and SLE.

Restriction of diverse retroviruses by SAMHD1

BackgroundSAMHD1 is a triphosphohydrolase that restricts the replication of HIV-1 and SIV in myeloid cells. In macrophages and dendritic cells, SAMHD1 restricts virus replication by diminishing the deoxynucleotide triphosphate pool to a level below that which supports lentiviral reverse transcription. HIV-2 and related SIVs encode the accessory protein Vpx to induce the proteasomal degradation of SAMHD1 following virus entry. While SAMHD1 has been shown to restrict HIV-1 and SIV, the breadth of its restriction is not known and whether other viruses have a means to counteract the restriction has not been determined.ResultsWe show that SAMHD1 restricts a wide array of divergent retroviruses, including the alpha, beta and gamma classes. Murine leukemia virus was restricted by SAMHD1 in macrophages yet removal of SAMHD1 did not alleviate the block to infection because of an additional block to viral nuclear import. Prototype foamy virus (PFV) and Human T cell leukemia virus type I (HTLV-1) were the only retroviruses tested that were not restricted by SAMHD1. PFV reverse transcribes predominantly prior to entry and thus is unaffected by the dNTP level in the target cell. It is possible that HTLV-1 has a mechanism to render the virus resistant to SAMHD1-mediated restriction.ConclusionThe results suggest that SAMHD1 has broad anti-retroviral activity against which most viruses have not found an escape.

Foamy Virus Biology and Its Application for Vector Development

Spuma- or foamy viruses (FV), endemic in most non-human primates, cats, cattle and horses, comprise a special type of retrovirus that has developed a replication strategy combining features of both retroviruses and hepadnaviruses. Unique features of FVs include an apparent apathogenicity in natural hosts as well as zoonotically infected humans, a reverse transcription of the packaged viral RNA genome late during viral replication resulting in an infectious DNA genome in released FV particles and a special particle release strategy depending capsid and glycoprotein coexpression and specific interaction between both components. In addition, particular features with respect to the integration profile into the host genomic DNA discriminate FV from orthoretroviruses. It appears that some inherent properties of FV vectors set them favorably apart from orthoretroviral vectors and ask for additional basic research on the viruses as well as on the application in Gene Therapy. This review will summarize the current knowledge of FV biology and the development as a gene transfer system.