Internal Promoter Research Articles

Alternative Splicing, Internal Promoter, Nonsense-Mediated Decay, or All Three: Explaining the Distribution of Truncation Variants in Titin.

Truncating mutations in the giant sarcomeric gene Titin are the most common type of genetic alteration in dilated cardiomyopathy. Detailed studies have amassed a wealth of information about truncating variant position in cases and controls. Nonetheless, considerable confusion exists as to how to interpret the pathogenicity of these variants, hindering our ability to make useful recommendations to patients. Building on our recent discovery of a conserved internal promoter within the Titin gene, we sought to develop an integrative statistical model to explain the observed pattern of Titin truncation variants in patients with dilated cardiomyopathy and population controls. We amassed Titin truncation mutation information from 1714 human dilated cardiomyopathy cases and >69 000 controls and found 3 factors explaining the distribution of Titin mutations: (1) alternative splicing, (2) whether the internal promoter Cronos isoform was disrupted, and (3) whether the distal C terminus was targeted (in keeping with the observation that truncation variants in this region escape nonsense-mediated decay and continue to be incorporated in the sarcomere). A model using these 3 factors had strong predictive performance with an area under the receiver operating characteristic curve of 0.81. Accordingly, individuals with either the most severe form of dilated cardiomyopathy or whose mutations demonstrated clear family segregation experienced the highest risk profile across all 3 components. We conclude that quantitative models derived from large-scale human genetic and phenotypic data can be applied to help overcome the ever-growing challenges of genetic data interpretation. Results of our approach can be found at http://cvri.ucsf.edu/~deo/TTNtruncationvariant.html.

The time-resolved transcriptome of C. elegans.

We generated detailed RNA-seq data for the nematode Caenorhabditis elegans with high temporal resolution in the embryo as well as representative samples from post-embryonic stages across the life cycle. The data reveal that early and late embryogenesis is accompanied by large numbers of genes changing expression, whereas fewer genes are changing in mid-embryogenesis. This lull in genes changing expression correlates with a period during which histone mRNAs produce almost 40% of the RNA-seq reads. We find evidence for many more splice junctions than are annotated in WormBase, with many of these suggesting alternative splice forms, often with differential usage over the life cycle. We annotated internal promoter usage in operons using SL1 and SL2 data. We also uncovered correlated transcriptional programs that span >80 kb. These data provide detailed annotation of the C. elegans transcriptome.

Abstract 64: An Integrated Model for Titin Truncation Mutation Interpretation

Background: We recently discovered a conserved internal promoter in the Titin gene, which explains why truncating mutations in the C-terminal two thirds of the zebrafish ttna protein result in more severe disease, recapitulating a puzzling observation in human dilated cardiomyopathy (DCM) patients. Here we focus on the contribution of alternative splicing to the DCM phenotype, both in zebrafish Titin truncation mutants and in the context of an integrative model for Titin mutation interpretation. Methods and Results: Using CRISPR/Cas9, we disrupted an alternatively spliced exon in the I-band of Titin , normally present in zebrafish heart but absent in skeletal muscle. The resulting mutants had, on average, a milder cardiac phenotype than those with mutations in constitutive exons but also showed striking inter-sibling variability in disease expression, ranging from intact cardiac blood flow to severe early demise. The mutant exon demonstrated nonsense-altered splicing and disease severity paralleled selective deficiency in Titin transcript level, implying that variability in mutated exon inclusion coupled with nonsense-mediated decay (NMD) modulated phenotype. We next amassed Titin mutation information from 1785 human DCM cases and >68,000 controls to model mutation distribution and found three variance components 1) splicing; 2) internal isoform disruption; and 3) targeting of the C-terminal 2000 amino acids. An integrated model demonstrated strong predictive performance with an area under the receiver operating characteristic curve of 0.79 and correctly identified the highest risk individuals. Conclusions: We conclude that genetically targeted models and large-scale human data can be complementary in overcoming the challenges of genetic data interpretation.

Important role of N108 residue in binding of bovine foamy virus transactivator Tas to viral promoters.

BackgroundBovine foamy virus (BFV) encodes the transactivator BTas, which enhances viral gene transcription by binding to the long terminal repeat promoter and the internal promoter. In this study, we investigated the different replication capacities of two similar BFV full-length DNA clones, pBS-BFV-Y and pBS-BFV-B.ResultsHere, functional analysis of several chimeric clones revealed a major role for the C-terminal region of the viral genome in causing this difference. Furthermore, BTas-B, which is located in this C-terminal region, exhibited a 20-fold higher transactivation activity than BTas-Y. Sequence alignment showed that these two sequences differ only at amino acid 108, with BTas-B containing N108 and BTas-Y containing D108 at this position. Results of mutagenesis studies demonstrated that residue N108 is important for BTas binding to viral promoters. In addition, the N108D mutation in pBS-BFV-B reduced the viral replication capacity by about 1.5-fold.ConclusionsOur results suggest that residue N108 is important for BTas binding to BFV promoters and has a major role in BFV replication. These findings not only advances our understanding of the transactivation mechanism of BTas, but they also highlight the importance of certain sequence polymorphisms in modulating the replication capacity of isolated BFV clones.

PRDM8 internal promoter hyperhydroxymethylation correlates with increased expression of the corresponding transcript in Downsyndrome.

Downsyndrome (DS) is the most common form of intellectual disability associated with central nervous system abnormalities and results from an extra complete or partial copy of human chromosome21. However, whether DNA hydroxymethylation is perturbed in a specific gene associated with DS phenotypes, or the alteration of DNA hydroxymethy-lation results in changes of gene expression in DS remains unidentified. The current study mapped 5‑methylcytosine and 5‑hydroxymethylcytosine at CpG islands of the PR domain containing8 (PRDM8) in the peripheral blood of 16DS and 19normal samples by oxidative bisulfite-pyrosequencing. Furthermore, the association of the expression levels of the two transcripts and epigenetic modification in different genomic contexts of PRDM8 was analyzed. The results demonstrated hypermethylation and hyperhydroxymethylation at the internal promoter of PRDM8 in DS, and significantly increased the expression of PRDM8 transcript variant2 in the DSpatients (median3.9vs.2.04; P=0.016), accompanied by a positive correlation between the expression of two PRDM8 transcripts and hydroxymethylation at the corresponding external and internal promoters in patients, although not in the controls. A similar association was observed between the expression of transcript variant1 and intragenic methylation of PRDM8. Taken together, the results of the present study suggest a critical role for DNA hydroxymethylation and methylation in regulating abnormal PRDM8 overexpression in DS.

The Structure-Specific Recognition Protein 1 Associates with Lens Epithelium-Derived Growth Factor Proteins and Modulates HIV-1 Replication

The lens epithelium-derived growth factor p75 (LEDGF/p75) is a chromatin-bound protein essential for efficient lentiviral integration. Genome-wide studies have located LEDGF/p75 inside actively transcribed genes where it mediates lentiviral integration. Although its role in HIV-1 integration is clearly established, the role of LEDGF/p75-associated proteins in HIV-1 infection remains unexplored. Using protein–protein interaction assays, we demonstrated that LEDGF/p75 complexes with a chromatin-remodeling complex facilitates chromatin transcription (FACT), a heterodimer of the structure-specific recognition protein 1 (SSRP1) and the human homolog of suppressor of Ty 16 (hSpt16). Detailed analysis of the interaction of LEDGF/p75 with the FACT complex indicates that LEDGF/p75 interacts with SSRP1 in an hSpt16-independent manner that requires the PWWP domain of LEDGF proteins and the HMG domain of SSRP1. Functional characterizations demonstrate a LEDGF/p75-independent role of SSRP1 in the regulation of HIV-1 replication. shRNA-mediated partial knockdown of SSRP1 reduces HIV-1 infection, but not Murine Leukemia Virus, in human CD4+ T cells. Similarly, SSRP1 knockdown affects infection by HIV-1-derived viruses that express genes from the viral LTR but not from an internal immediate-early CMV promoter, suggesting a role of SSRP1 in LTR-driven gene expression but not in viral DNA integration. Together, our data demonstrate for the first time the association of LEDGF proteins with the FACT complex and give further support to a role of SSRP1 in HIV-1 infection.

Possible interaction between the bacterial transcription factor ArtA and the eukaryotic RNA polymerase III promoter.

Eukaryotic RNA polymerase III (RNAP III) transcribes tRNA genes and short interspersed elements that have internal promoters consisting of A- and B-blocks. The B-block binding subunit of the transcription initiation factor TFIIIC binds to the B-block. The mobile bacterial insertion sequence (IS) 1 contains a RNAP III promoter-like sequence, which stimulates bacterial transcription along with the bacterial ArtA protein. Here, the DNA-binding ability of ArtA was examined in vitro using a simple, newly developed method. Various DNA fragments, including RNAP III promoter fragments, were separately incubated with purified ArtA, and then loaded onto a polyacrylamide gel. Since DNAs bound by ArtA remain in the gel wells during electrophoresis, SDS was added into the wells at the electrophoresis halfway point. It was hypothesized that SDS would dissociate the DNA-ArtA complexes in the wells, and then the DNAs would begin to migrate. In fact, new bands appeared in all of the lanes at similar intensities, indicating that ArtA binds nonspecifically to DNA. Therefore, labeled wild-type RNAP III promoter fragments were incubated with either the unlabeled wild-type or mutant fragments and ArtA, and electrophoresed. The B-block(-like) sequences of IS1, a human Alu element, and an anuran tRNA gene were important for binding to ArtA. Additionally, in silico analyses revealed the presence of the RNAP III promoter-like structures in the IS1 isoforms and the IS3 family elements. These results suggest the presence of parts of the RNAP III transcription machinery in bacteria, and might imply that its prototype existed in the common ancestor.

Mechanism of Antiactivation at the Pseudomonas sp. Strain ADP σN-Dependent PatzT Promoter.

PatzT is an internal promoter of the atzRSTUVW operon that directs the synthesis of AtzT, AtzU, AtzV, and AtzW, components of an ABC-type cyanuric acid transport system. PatzT is σ(N) dependent, activated by the general nitrogen control regulator NtrC with the assistance of protein integration host factor (IHF), and repressed by the LysR-type transcriptional regulator (LTTR) AtzR. We have used a variety of in vivo and in vitro gene expression and protein-DNA interaction assays to assess the mechanisms underlying AtzR-dependent repression of PatzT Here, we show that repression only occurs when AtzR and NtrC interact simultaneously with the PatzT promoter region, indicating that AtzR acts as an antiactivator to antagonize activation by NtrC. Furthermore, repression requires precise rotational orientation of the AtzR and NtrC binding sites, strongly suggesting protein-protein interaction between the two proteins on the promoter region. Further exploration of the antiactivation mechanism showed that although AtzR-dependent repression occurs prior to open complex formation, AtzR does not alter the oligomerization state of NtrC or inhibit NtrC ATPase activity when bound to the PatzT promoter region. Taken together, these results strongly suggest that PatzT-bound AtzR interacts with NtrC to prevent the coupling of NtrC-mediated ATP hydrolysis with the remodeling of the interactions between E-σ(N) and PatzT that lead to open complex formation. Here, we describe a unique mechanism by which the regulatory protein AtzR prevents the activation of the σ(N)-dependent promoter PatzT Promoters of this family are always positively regulated, but there are a few examples of overlapping negative regulation. The mechanism described here is highly unconventional and involves an interaction between the repressor and activator proteins to prevent the action of the repressor protein on the RNA polymerase-promoter complex.

687. Development of a Safe and Effective Combinatorial Foamy Virus Vector for HIV Gene Therapy

The Berlin patient and successes in retroviral hematopoietic stem cell (HSC) gene therapy suggest that gene therapy may provide a functional cure for HIV. However, retroviral-HSC gene therapy has resulted in serious adverse side effects due to vector mediated genotoxicity, including leukemia. Foamy virus (FV) vectors have a promising integration profile and may be safer than retroviral vectors. FV vectors also can efficiently deliver anti-HIV transgenes that can reduce the titer of HIV-1 based lentiviral vectors. We report a novel combinatorial anti-HIV FV vector that uses a housekeeping elongation factor 1 alpha (EF1α) promoter but still potently blocks HIV infection. We first evaluated the relative potency of various previously described anti-HIV transgenes, including the C46 fusion inhibitor, the F12-Vif derivative Chim3, lens epithelium derived growth factor-integrase binding domain (LIBD) and TRIM5α-CyclophilinA fusion (TCypA) in a FV vector background. We found that C46 was the most potent anti-HIV transgene, followed by TCypA and LEDGF-IBD. Next, we hypothesized that using a less-genotoxic internal promoter to drive the transgenes would reduce vector-mediated genotoxicity. Therefore, a direct comparison was made between the efficacy of housekeeping gene promoters (EF1α and Ubiquitin C; UbC) and a highly genotoxic SFFV promoter in expressing the C46 transgene and subsequently blocking HIV replication. We observed that C46 EF1α had ~2 to 4 fold higher anti-HIV effect than C46 driven by either SFFV or UbC promoters, respectively. Based on these results, we designed a novel combinatorial FV vector expressing three anti-HIV transgenes: C46, TcypA and LIBD. The gene cassette was driven by an EF1α promoter and also has mCherry as a reporter gene (FV-E C46TLmC-W). This vector can be produced at high titer, 1.4 × 107 transducing units/ml which is critical for clinical translation. This novel combinatorial anti-HIV FV vector showed a higher potency in blocking HIV replication than C46 alone at a late time point, 21 days post HIV infection (Figure 1Figure 1). An in vitro competitive survival advantage assay indicated that cells transduced with our novel combinatorial anti-HIV FV vectors are highly resistant to HIV infection compared to cells transduced with FV expressing a control EGFP reporter gene alone (FV-EG-W) (Figure 2Figure 2). Further studies will be focused on the efficacy of our novel combinatorial anti-HIV FV vector in human CD34+ cells transplanted in a mouse xenotransplant in vivo model. Our goal is to develop a safe and potent combinatorial FV vector for clinical studies.View Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint Slide

N-terminally truncated FOXP1 protein expression and alternate internal FOXP1 promoter usage in normal and malignant B cells.

Strong FOXP1 protein expression is a poor risk factor in diffuse large B-cell lymphoma and has been linked to an activated B-cell-like subtype, which preferentially expresses short FOXP1 (FOXP1S) proteins. However, both short isoform generation and function are incompletely understood. Here we prove by mass spectrometry and N-terminal antibody staining that FOXP1S proteins in activated B-cell-like diffuse large B-cell lymphoma are N-terminally truncated. Furthermore, a rare strongly FOXP1-expressing population of normal germinal center B cells lacking the N-terminus of the regular long protein (FOXP1L) was identified. Exon-targeted silencing and transcript analyses identified three alternate 5' non-coding exons [FOXP1-Ex6b(s), FOXP1-Ex7b and FOXP1-Ex7c], downstream of at least two predicted promoters, giving rise to FOXP1S proteins. These were differentially controlled by B-cell activation and methylation, conserved in murine lymphoma cells, and significantly correlated with FOXP1S protein expression in primary diffuse large B-cell lymphoma samples. Alternatively spliced isoforms lacking exon 9 (e.g. isoform 3) did not encode FOXP1S, and an alternate long human FOXP1 protein (FOXP1AL) likely generated from a FOXP1-Ex6b(L) transcript was detected. The ratio of FOXP1L:FOXP1S isoforms correlated with differential expression of plasmacytic differentiation markers in U-2932 subpopulations, and altering this ratio was sufficient to modulate CD19 expression in diffuse large B-cell lymphoma cell lines. Thus, the activity of multiple alternate FOXP1 promoters to produce multiple protein isoforms is likely to regulate B-cell maturation.

Constitutive Expression of a Nag-Like Dioxygenase Gene through an Internal Promoter in the 2-Chloronitrobenzene Catabolism Gene Cluster of Pseudomonas stutzeri ZWLR2-1.

The gene cluster encoding the 2-chloronitrobenzene (2CNB) catabolism pathway in Pseudomonas stutzeri ZWLR2-1 is a patchwork assembly of a Nag-like dioxygenase (dioxygenase belonging to the naphthalene dioxygenase NagAaAbAcAd family from Ralstonia sp. strain U2) gene cluster and a chlorocatechol catabolism cluster. However, the transcriptional regulator gene usually present in the Nag-like dioxygenase gene cluster is missing, leaving it unclear how this cluster is expressed. The pattern of expression of the 2CNB catabolism cluster was investigated here. The results demonstrate that the expression was constitutive and not induced by its substrate 2CNB or salicylate, the usual inducer of expression in the Nag-like dioxygenase family. Reverse transcription-PCR indicated the presence of at least one transcript containing all the structural genes for 2CNB degradation. Among the three promoters verified in the gene cluster, P1 served as the promoter for the entire catabolism operon, but the internal promoters P2 and P3 also enhanced the transcription of the genes downstream. The P3 promoter, which was not previously defined as a promoter sequence, was the strongest of these three promoters. It drove the expression of cnbAcAd encoding the dioxygenase that catalyzes the initial reaction in the 2CNB catabolism pathway. Bioinformatics and mutation analyses suggested that this P3 promoter evolved through the duplication of an 18-bp fragment and introduction of an extra 132-bp fragment. The release of many synthetic compounds into the environment places selective pressure on bacteria to develop their ability to utilize these chemicals to grow. One of the problems that a bacterium must surmount is to evolve a regulatory device for expression of the corresponding catabolism genes. Considering that 2CNB is a xenobiotic that has existed only since the onset of synthetic chemistry, it may be a good example for studying the molecular mechanisms underlying rapid evolution in regulatory networks for the catabolism of synthetic compounds. The 2CNB utilizer Pseudomonas stutzeri ZWLR2-1 in this study has adapted itself to the new pollutant by evolving the always-inducible Nag-like dioxygenase into a constitutively expressed enzyme, and its expression has escaped the influence of salicylate. This may facilitate an understanding of how bacteria can rapidly adapt to the new synthetic compounds by evolving its expression system for key enzymes involved in the degradation of a xenobiotic.

Oncogenic Intra-p53 Family Member Interactions in Human Cancers.

The p53 gene family members p53, p73, and p63 display several isoforms derived from the presence of internal promoters and alternative splicing events. They are structural homologs but hold peculiar functional properties. p53, p73, and p63 are tumor suppressor genes that promote differentiation, senescence, and apoptosis. p53, unlike p73 and p63, is frequently mutated in cancer often displaying oncogenic “gain of function” activities correlated with the induction of proliferation, invasion, chemoresistance, and genomic instability in cancer cells. These oncogenic functions are promoted either by the aberrant transcriptional cooperation of mutant p53 (mutp53) with transcription cofactors (e.g., NF-Y, E2F1, Vitamin D Receptor, Ets-1, NF-kB and YAP) or by the interaction with the p53 family members, p73 and p63, determining their functional inactivation. The instauration of these aberrant transcriptional networks leads to increased cell growth, low activation of DNA damage response pathways (DNA damage response and DNA double-strand breaks response), enhanced invasion, and high chemoresistance to different conventional chemotherapeutic treatments. Several studies have clearly shown that different cancers harboring mutant p53 proteins exhibit a poor prognosis when compared to those carrying wild-type p53 (wt-p53) protein. The interference of mutantp53/p73 and/or mutantp53/p63 interactions, thereby restoring p53, p73, and p63 tumor suppression functions, could be among the potential therapeutic strategies for the treatment of mutant p53 human cancers.

Lysine residues K66, K109, and K110 in the bovine foamy virus transactivator protein are required for transactivation and viral replication.

Bovine foamy virus (BFV) is a complex retrovirus that infects cattle. Like all retroviruses, BFV encodes a transactivator Tas protein (BTas) that increases gene transcription from viral promoters. BFV encodes two promoters that can interact with BTas, a conserved promoter in the 5' long terminal repeat (LTR) and a unique internal promoter (IP). Our previous study showed that BTas is acetylated by p300 at residues K66, K109, and K110, which markedly enhanced the ability of BTas to bind to DNA. However, whether these residues are important for BFV replication was not determined. Therefore, in this study we provide direct evidence that BTas is required for BFV replication and demonstrate that residues K66, K109, and K110 are critical for BTas function and BFV replication. Full-length infectious clones were generated, which were BTas deficient or contained lysine to arginine (K→R) mutations at position 66, 109, and/or 110. In vivo data indicated that K→R mutations at positions 66, 109, and 110 in BTas impaired transactivation of both the LTR and IP promoters. In addition, the K→R mutations in full-length infectious clones reduced expression of viral proteins, and the triple mutant and BTas deletion completely abrogated viral replication. Taken together, these results indicate that lysine residues at positions 66, 109, and 110 in the BTas protein are crucial for BFV replication and suggest a potential role for BTas acetylation in regulating the viral life cycle.

Insulated Foamy Viral Vectors.

Retroviral vector-mediated gene therapy is promising, but genotoxicity has limited its use in the clinic. Genotoxicity is highly dependent on the retroviral vector used, and foamy viral (FV) vectors appear relatively safe. However, internal promoters may still potentially activate nearby genes. We developed insulated FV vectors, using four previously described insulators: a version of the well-studied chicken hypersensitivity site 4 insulator (650cHS4), two synthetic CCCTC-binding factor (CTCF)-based insulators, and an insulator based on the CCAAT box-binding transcription factor/nuclear factor I (7xCTF/NF1). We directly compared these insulators for enhancer-blocking activity, effect on FV vector titer, and fidelity of transfer to both proviral long terminal repeats. The synthetic CTCF-based insulators had the strongest insulating activity, but reduced titers significantly. The 7xCTF/NF1 insulator did not reduce titers but had weak insulating activity. The 650cHS4-insulated FV vector was identified as the overall most promising vector. Uninsulated and 650cHS4-insulated FV vectors were both significantly less genotoxic than gammaretroviral vectors. Integration sites were evaluated in cord blood CD34(+) cells and the 650cHS4-insulated FV vector had fewer hotspots compared with an uninsulated FV vector. These data suggest that insulated FV vectors are promising for hematopoietic stem cell gene therapy.

DNA methylation in PRDM8 is indicative for dyskeratosis congenita.

Dyskeratosis congenita (DKC) is associated with impaired telomere maintenance and with clinical features of premature aging. In this study, we analysed global DNA methylation (DNAm) profiles of DKC patients. Age-associated DNAm changes were not generally accelerated in DKC, but there were significant differences to DNAm patterns of healthy controls, particularly in CpG sites related to an internal promoter region of PR domain containing 8 (PRDM8). Notably, the same genomic region was also hypermethylated in aplastic anemia (AA) – another bone marrow failure syndrome. Site-specific analysis of DNAm level in PRDM8 with pyrosequencing and MassARRAY validated aberrant hypermethylation in 11 DKC patients and 27 AA patients. Telomere length, measured by flow-FISH, did not directly correlate with DNAm in PRDM8. Therefore the two methods may be complementary to also identify patients with still normal telomere length. In conclusion, blood of DKC patients reveals aberrant DNAm patterns, albeit age-associated DNAm patterns are not generally accelerated. Aberrant hypermethylation is particularly observed in PRDM8 and this may support identification and classification of bone marrow failure syndromes.

Immunoreactions for P53 isoforms are associated with ultrastructural proliferative profiles in benign thyroid nodules.

P53 isoforms originate from the alternative initiation of P53 gene translation through usage of an internal promoter located in intron 4. All P53 isoforms are spliced in intron 9 and may modulate cell proliferation and cell fate outcome in response to DNA damage. To examine immunoexpression of P53 isoforms in benign proliferative lesions occurring in multinodular thyroids and to assess the ultrastructural phenotype of P53 distribution in the thyrocytes of those lesions by electron microscopy. By immunohistochemistry and transmission electron microscopy (TEM), we evaluated 38 multinodular thyroids containing a total of 102 benign lesions: 38 nodular goiters (NG; colloid=20, parenchymatous=18), 52 follicular adenomas (FA) and 12 Hashimoto's thyroditis (HT). FA were classified into 10 normo-follicular, 9 macro-follicular, 28 micro-follicular and 5 solid variants. Immunoreaction for P53 isoforms was observed in approximately 50% of all lesions, except macrofollicular variant FA (33%). At TEM analysis, immunoreactive NG, FA and TH lesions showed signs of proliferation by simultaneous appearance of dispersed chromatin, increased amounts of cytoplasmic organelles and dilation of the rough endoplasmic reticulum. TEM signs of apoptosis and proliferation were also detected in FA, but with different rates compared to NG. The immunohistochemical expression of P53 isoforms in NG, FA and HT suggests their role in the development of these lesions. Ultrastructural findings support the hypothesis that P53 immunoexpression correlates with reactive proliferative changes in thyrocytes.

Modus operandi of SMEs towards cooperative innovation: evidence from Bizkaia

In recent times, open innovation has become fashionable as a way to make up for limited internal efforts around innovation. In the present paper, we review the state of cooperative innovation among small- and medium-sized companies (SMEs) by categorising their modus operandi regarding innovation in general and towards cooperative innovation in particular. To that end, we create a framework for analysis that is rooted in the dichotomy between doing-using-interacting (DUI) and science-technology-investigation (STI). The inventory obtained serves as a basis for identifying implications for managers with regard to enhancing the propensity for cooperative innovation among SMEs and increasing the benefits that can follow from it. We find that SMEs should devote particularly care to creating a position for an internal innovation promoter to act as a boundary spanner between the firm and external partners. Additional recommendations for thought and action are to look beyond the regional home base for innovation partners and consider implementing more formal governance arrangements for cooperative innovation.

Dystrophin Dp71, a novel tumor suppressor?

Dystrophins belong to a group of proteins encoding by the Duchenne muscular dystrophy ( DMD ) gene. DMD gene generates full-length dystrophin (427 kDa) and different C-terminal truncated isoforms through alternative usage of internal promoters. Short isoforms are termed in accordance with their molecular mass as Dp140, Dp116, Dp71 and Dp40. Dystrophin Dp71, is ubiquitously expresses, with the exception of skeletal muscle, and is the predominant DMD gene product in nervous system. In addition, Dp71 undergoes alternative splicing of exons 71 and/or 78 to generate different splicing variants, being Dp71f (−71/−78) and Dp71d (−71/+78) the most studied isoforms. While deficiency of dystrophin causes muscle degeneration, absence of Dp71 has been implicated in cognitive impairment and abnormal retinal physiology, two well-defined non-muscular alterations of DMD patients, reviewed in (1). As dystrophin does, Dp71 associates with sarcoglycans, dystroglycans, syntrophins and dystrobrevin, to conform the plasma membrane-associated dystrophin associated protein complex. Such protein assembly provides stability to the plasma membrane and modulates cell signaling. Interestingly, Dp71 undergoes nuclear import trough recognition of an atypical nuclear localization signal by the importin α2/β1 system. Nuclear Dp71 in turn, is involved in the maintenance of nuclear architecture, via its interaction with nuclear envelope proteins, including emerin and lamins A/C and B1 (2). Thus, it appears that Dp71 is a multifunctional protein that interacts with different partners in both the cytoplasm and nucleus, which enables it to modulate a variety of cell functions.

TauCstF-64 Mediates Correct mRNA Polyadenylation and Splicing of Activator and Repressor Isoforms of the Cyclic AMP-Responsive Element Modulator (CREM) in Mouse Testis.

Spermatogenesis is coordinated by the spatial and temporal expression of many transcriptional and posttranscriptional factors. The cyclic AMP-responsive element modulator (CREM) gene encodes both activator and repressor isoforms that act as transcription factors to regulate spermiogenesis. We found that the testis-expressed paralog of CstF-64, tauCstF-64 (gene symbol Cstf2t), is involved in a polyadenylation site choice switch of Crem mRNA and leads to an overall decrease of the Crem mRNAs that are generated from internal promoters in Cstf2t−/− mice. More surprisingly, loss of tauCstF-64 also leads to alternative splicing of Crem exon 4, which contains an important activation domain. Thus, testis-specific CREMtau2 isoform protein levels are reduced in Cstf2t−/− mice. Consequently, expression of 15 CREM-regulated genes is decreased in testes of Cstf2t−/− mice at 25 days postpartum. These effects might further contribute to the infertility phenotype of these animals. This demonstrates that tauCstF-64 is an important stage-specific regulator of Crem mRNA processing that modulates the spatial and temporal expression of downstream stage-specific genes necessary for the proper development of sperm in mice.

Preclinical Development and Validation of a Hematopoietic Stem Cell-Based Lentivector Treatment for Hemophilia a

Hemophilia A is a monogenic bleeding disorder currently treated by lifelong infusions of factor VIII (fVIII). The development of gene-based treatments for hemophilia A has been hampered by several biological hurdles including the low level expression of fVIII from genetically engineered target cells, the large transgene size, and transgene product immunogenicity. We now show that these major hurdles are overcome by i) using a bioengineered FVIII transgene (ET3) that expresses at 10 - 100-fold higher levels compared to other human FVIII sequences, ii) targeting hematopoietic stem cells (HSC) ex vivo using a self-inactivating (SIN) lentiviral vector (LV) encoding ET3, and iii) using a non-myeloablative conditioning regimen prior to HSC transplantation that results in transgene product associated immune tolerance. The clinical trial design and preclinical data were favorably reviewed by the RAC, Institutional Biosafety Committee, and FDA. However upon full-scale clinical production of the SIN-ET3-LV, an additional hurdle was discovered that resulted in low LV manufacturing titer, which we showed is due to promoter competition between the CMV/HIV-LTR and the internal promoter driving ET3 production (eg. EF1a). We show that high-titer recombinant LV encoding ET-3 can be achieved by replacing the EF1a internal promoter with one that has low activity in the 293T producer cells. CD68 is a monocyte-restricted promoter that is not active in 293T producer cell lines and thus minimizes promoter interference. Using a SIN-CD68-ET3-LV expression vector, high titer virus was generated using a laboratory scale production strategy as well as a 6 L scalable clinical LV production platform. Our scalable manufacturing process generated titers >2 x 109/ml, which is sufficient to support an ex vivo HSC transduction/transplantation pilot/phase I clinical trial. Late stage preclinical testing using clinically processed vector showed that the high titer virus efficiently transduces hematopoietic cell lines, as determined by quantitative PCR, and Southern analysis, which confirmed integrated transgene stability over 8 weeks. Efficient and stable gene transfer into murine Sca-1+ cells harvested from hemophilia A mice was also achieved. A dose response of LV addition to Sca-1 cells and FVIII expression was observed in transplanted mice. Expression of >40% normal FVIII levels was achieved at multiplicity of infections (MOIs) as low 20, and long term FVIII expression was observed post HSCT. No difference in the engraftment of genetically modified cells as compared to mock-modified cells was observed. In addition, efficient, stable and dose responsive gene marking (up to 0.6 VCN/cell) of human mobilized peripheral blood(mPB)-derived CD34+ cells was reproducibly achieved. Transduction of mPB-CD34+ cells at MOIs ranging from 1 to 125 had no effect on methylcellulose colony forming unit growth when comparing SIN-CD68-ET3-LV to non-modified cells. In addition, there was no effect on mPB-CD34+ cell engraftment of NSG mice when comparing modified and mock modified cells. Furthermore, next generation sequencing-based insertion site analysis showed integration of SIN-CD68-ET3-LV to be similar, with respect to integration events near oncogenes, to that of other vectors in current clinical trials. Collectively, this comprehensive, preclinical data set provides support for regulatory submissions to conduct a pilot/phase I clinical trial of a SIN-CD68-ET3-LV transduced-HSC transplantation product for the treatment of hemophilia A. DisclosuresDoering:Bayer Healthcare: Consultancy, Honoraria, Research Funding; Expression Therapeutics: Equity Ownership. Denning:Expression Therapeutics: Employment. Spencer:Expression Therapeutics: Equity Ownership.