A replicating LCMV-based vaccine for the treatment of solid tumors.

Harnessing the immune system to eradicate tumors requires identification and targeting of tumor antigens, including tumor-specific neoantigens and tumor-associated self-antigens. Tumor-associated antigens are subject to existing immune tolerance, which must be overcome by immunotherapies. Despite many novel immunotherapies reaching clinical trials, inducing self-antigen-specific immune responses remains challenging. Here, we systematically investigate viral-vector-based cancer vaccines encoding a tumor-associated self-antigen (TRP2) for the treatment of established melanomas in preclinical mouse models, alone or in combination with adoptive T cell therapy. We reveal that, unlike foreign antigens, tumor-associated antigens require replication of lymphocytic choriomeningitis virus (LCMV)-based vectors to break tolerance and induce effective antigen-specific CD8+ T cell responses. Immunization with a replicating LCMV vector leads to complete tumor rejection when combined with adoptive TRP2-specific T cell transfer. Importantly, immunization with replicating vectors leads to extended antigen persistence in secondary lymphoid organs, resulting in efficient T cell priming, which renders previously "cold" tumors open to immune infiltration and reprograms the tumor microenvironment to "hot." Our findings have important implications for the design of next-generation immunotherapies targeting solid cancers utilizing viral vectors and adoptive cell transfer.

Heterologous DNA-prime/protein-boost immunization with a monomeric SARS-CoV-2 spike antigen redundantizes the trimeric receptor-binding domain structure to induce neutralizing antibodies in old mice.

A multitude of alterations in the old immune system impair its functional integrity. Closely related, older individuals show, for example, a reduced responsiveness to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccines. However, systematic strategies to specifically improve the efficacy of vaccines in the old are missing or limited to simple approaches like increasing the antigen concentration or injection frequencies. We here asked whether the intrinsic, trimeric structure of the SARS-CoV-2 spike (S) antigen and/or a DNA- or protein-based antigen delivery platform affects priming of functional antibody responses particularly in old mice. The used S-antigens were primarily defined by the presence/absence of the membrane-anchoring TM domain and the closely interlinked formation/non-formation of a trimeric structure of the receptor binding domain (S-RBD). Among others, we generated vectors expressing prefusion-stabilized, cell-associated (TM+) trimeric "S2-P" or secreted (TM-) monomeric "S6-PΔTM" antigens. These proteins were produced from vector-transfected HEK-293T cells under mild conditions by Strep-tag purification, revealing that cell-associated but not secreted S proteins tightly bound Hsp73 and Grp78 chaperones. We showed that both, TM-deficient S6-PΔTM and full-length S2-P antigens elicited very similar S-RBD-specific antibody titers and pseudovirus neutralization activities in young (2-3 months) mice through homologous DNA-prime/DNA-boost or protein-prime/protein-boost vaccination. The trimeric S2-P antigen induced high S-RBD-specific antibody responses in old (23-24 months) mice through DNA-prime/DNA-boost vaccination. Unexpectedly, the monomeric S6-PΔTM antigen induced very low S-RBD-specific antibody titers in old mice through homologous DNA-prime/DNA-boost or protein-prime/protein-boost vaccination. However, old mice efficiently elicited an S-RBD-specific antibody response after heterologous DNA-prime/protein-boost immunization with the S6-PΔTM antigen, and antibody titers even reached similar levels and neutralizing activities as in young mice and also cross-reacted with different S-variants of concern. The old immune system thus distinguished between trimeric and monomeric S protein conformations: it remained antigen responsive to the trimeric S2-P antigen, and a simple change in the vaccine delivery regimen was sufficient to unleash its reactivity to the monomeric S6-PΔTM antigen. This clearly shows that both the antigen structure and the delivery platform are crucial to efficiently prime humoral immune responses in old mice and might be relevant for designing "age-adapted" vaccine strategies.

Human Monocytes Are Suitable Carriers for the Delivery of Oncolytic Herpes Simplex Virus Type 1 In Vitro and in a Chicken Embryo Chorioallantoic Membrane Model of Cancer.

Oncolytic viruses (OVs) are promising therapeutics for tumors with a poor prognosis. An OV based on herpes simplex virus type 1 (oHSV-1), talimogene laherparepvec (T-VEC), has been recently approved by the Food and Drug Administration (FDA) and by the European Medicines Agency (EMA) for the treatment of unresectable melanoma. T-VEC, like most OVs, is administered via intratumoral injection, underlining the unresolved problem of the systemic delivery of the oncolytic agent for the treatment of metastases and deep-seated tumors. To address this drawback, cells with a tropism for tumors can be loaded ex vivo with OVs and used as carriers for systemic oncolytic virotherapy. Here, we evaluated human monocytes as carrier cells for a prototype oHSV-1 with a similar genetic backbone as T-VEC. Many tumors specifically recruit monocytes from the bloodstream, and autologous monocytes can be obtained from peripheral blood. We demonstrate here that oHSV-1-loaded primary human monocytes migrated in vitro towards epithelial cancer cells of different origin. Moreover, human monocytic leukemia cells selectively delivered oHSV-1 to human head-and-neck xenograft tumors grown on the chorioallantoic membrane (CAM) of fertilized chicken eggs after intravascular injection. Thus, our work shows that monocytes are promising carriers for the delivery of oHSV-1s in vivo, deserving further investigation in animal models.

Evaluation of Human Mesenchymal Stromal Cells as Carriers for the Delivery of Oncolytic HAdV-5 to Head and Neck Squamous Cell Carcinomas.

Human multipotent mesenchymal stromal cells (hMSCs) are of significant therapeutic interest due to their ability to deliver oncolytic adenoviruses to tumors. This approach is also investigated for targeting head and neck squamous cell carcinomas (HNSCCs). HAdV-5-HexPos3, a recently reported capsid-modified vector based on human adenovirus type 5 (HAdV-5), showed strongly improved infection of both hMSCs and the HNSCC cell line UM-SCC-11B. Given that, we generated life cycle-unmodified and -modified replication-competent HAdV-5-HexPos3 vector variants and analyzed their replication within bone marrow- and adipose tissue-derived hMSCs. Efficient replication was detected for both life cycle-unmodified and -modified vectors. Moreover, we analyzed the migration of vector-carrying hMSCs toward different HNSCCs. Although migration of hMSCs to HNSCC cell lines was confirmed in vitro, no homing of hMSCs to HNSCC xenografts was observed in vivo in mice and in ovo in a chorioallantoic membrane model. Taken together, our data suggest that HAdV-5-HexPos3 is a potent candidate for hMSC-based oncolytic therapy of HNSCCs. However, it also emphasizes the importance of generating optimized in vivo models for the evaluation of hMSC as carrier cells.

Affilin-based retargeting of adenoviral vectors to the epidermal growth factor receptor.

INTRODUCTION: Treatment of head and neck squamous cell carcinomas (HNSCC) by oncolytic adenoviral vectors holds promise as an efficient anti-cancer therapy. The epidermal growth factor receptor (EGFR) represents an attractive target receptor since it is frequently overexpressed in many types of HNSCC. METHODS: To achieve EGFR-specific targeting by human adenovirus type 5 (HAdV-5) based vectors, the EGFR affinity ligand Affilin was covalently attached in a position specific manner either to the fiber or the hexon protein of the vector capsid. In vitro and in vivo studies investigated EGFR-specific cancer cell transduction, susceptibility to natural sequestration mechanisms, pharmacokinetics and biodistribution profiles of Affilin-decorated vectors. RESULTS: Affilin-decorated vectors showed strongly enhanced and EGFR-specific cancer cell transduction in vitro and less susceptibility to known sequestration mechanisms of HAdV-5 particles. However, in vivo neither systemic nor intratumoral vector administration resulted in an improved transduction of EGFR-positive tumors. Comprehensive analyses indicated hampered EGFR-targeting by Affilin-decorated vectors was caused by rapid vector particle consumption due to binding to the murine EGFR, insufficient tumor vascularization and poor target accessibility for Affilin in the solid tumor caused by a pronounced tumor stroma. CONCLUSION: In vitro studies yielded proof-of-concept results demonstrating that covalent attachment of a receptor-specific Affilin to the adenoviral capsid provides an effective and versatile tool to address cancer-specific target receptors by adenoviral vectors. Regarding EGFR as the vector target, off-target tissue transduction and low receptor accessibility within the tumor tissue prevented efficient tumor transduction by Affilin-decorated vectors, rendering EGFR a difficult-to-target receptor for adenoviral vectors.

Process- and product-related impurities in the ChAdOx1 nCov-19 vaccine.

ChAdOx1 nCov-19 and Ad26.COV2.S are approved vaccines inducing protective immunity against SARS-CoV-2 infection in humans by expressing the Spike protein of SARS-CoV-2. We analyzed protein content and protein composition of ChAdOx1 nCov-19 and Ad26.COV2.S by biochemical methods and by mass spectrometry. Four out of four tested lots of ChAdOx1 nCoV-19 contained significantly higher than expected levels of host cell proteins (HCPs) and of free viral proteins. The most abundant contaminating HCPs belonged to the heat-shock protein and cytoskeletal protein families. The HCP content exceeded the 400 ng specification limit per vaccine dose, as set by the European Medicines Agency (EMA) for this vaccine, by at least 25-fold and the manufacturer's batch-release data in some of the lots by several hundred-fold. In contrast, three tested lots of the Ad26.COV2.S vaccine contained only very low amounts of HCPs. As shown for Ad26.COV2.S production of clinical grade adenovirus vaccines of high purity is feasible at an industrial scale. Correspondingly, purification procedures of the ChAdOx1 nCov-19 vaccine should be modified to remove protein impurities as good as possible. Our data also indicate that standard quality assays, as they are used in the manufacturing of proteins, have to be adapted for vectored vaccines.

Huntingtin and Its Partner Huntingtin-Associated Protein 40: Structural and Functional Considerations in Health and Disease.

Since the discovery of the mutation causing Huntington's disease (HD) in 1993, it has been debated whether an expanded polyglutamine (polyQ) stretch affects the properties of the huntingtin (HTT) protein and thus contributes to the pathological mechanisms responsible for HD. Here we review the current knowledge about the structure of HTT, alone (apo-HTT) or in a complex with Huntingtin-Associated Protein 40 (HAP40), the influence of polyQ-length variation on apo-HTT and the HTT-HAP40 complex, and the biology of HAP40. Phylogenetic analyses suggest that HAP40 performs essential functions. Highlighting the relevance of its interaction with HTT, HAP40 is one of the most abundant partners copurifying with HTT and is rapidly degraded, when HTT levels are reduced. As the levels of both proteins decrease during disease progression, HAP40 could also be a biomarker for HD. Whether declining HAP40 levels contribute to disease etiology is an open question. Structural studies have shown that the conformation of apo-HTT is less constrained but resembles that adopted in the HTT-HAP40 complex, which is exceptionally stable because of extensive interactions between HAP40 and the three domains of HTT. The complex- and to some extent apo-HTT- resists fragmentation after limited proteolysis. Unresolved regions of apo-HTT, constituting about 25% of the protein, are the main sites of post-translational modifications and likely have major regulatory functions. PolyQ elongation does not substantially alter the structure of HTT, alone or when associated with HAP40. Particularly, polyQ above the disease length threshold does not induce drastic conformational changes in full-length HTT. Therefore, models of HD pathogenesis stating that polyQ expansion drastically alters HTT properties should be reconsidered.

Hexon modification of human adenovirus type 5 vectors enables efficient transduction of human multipotent mesenchymal stromal cells.

In adenovirus type 5 (HAdV-5)-derived viral vectors, the fiber protein has been the preferred locale for modifications to alter the natural viral tropism. Hexon, the most abundant capsid protein, has rarely been used for retargeting purposes, likely because the insertion of larger targeting peptides into Hexon often interferes with the assembly of the viral capsid. We previously observed that positively charged molecules enhance the transduction of human multipotent mesenchymal stromal cells (hMSCs)-a cell type of significant interest for clinical development but inefficiently transduced by unmodified HAdV-5-based vectors. As efficient HAdV-5-mediated gene transfer would greatly increase the therapeutic potential of hMSCs, we tested the hypothesis that introducing positively charged amino acids into Hexon might enhance the transduction of hMSCs, enabling efficient expression of selected transgenes. From the constructs that could be rescued as functional virions, one (HAdV-5-HexPos3) showed striking transduction of hMSCs with up to 500-fold increased efficiency. Evaluation of the underlying mechanism identified heparan sulfate proteoglycans (HSPGs) to be essential for virus uptake by the cells. The ease and efficiency of transduction of hMSCs with this vector will facilitate the development of genetically modified hMSCs as therapeutic vehicles in different disciplines, including oncology or regenerative medicine.

Vaccine-induced COVID-19 mimicry syndrome.

To fight the COVID-19 pandemic caused by the RNA virus SARS-CoV-2, a global vaccination campaign is in progress to achieve the immunization of billions of people mainly with adenoviral vector- or mRNA-based vaccines, all of which encode the SARS-CoV-2 Spike protein. In some rare cases, cerebral venous sinus thromboses (CVST) have been reported as a severe side effect occurring 4-14 days after the first vaccination and were often accompanied by thrombocytopenia. Besides CVST, splanchnic vein thromboses (SVT) and other thromboembolic events have been observed. These events only occurred following vaccination with adenoviral vector-based vaccines but not following vaccination with mRNA-based vaccines. Meanwhile, scientists have proposed an immune-based pathomechanism and the condition has been coined vaccine-induced immune thrombotic thrombocytopenia (VITT). Here, we describe an unexpected mechanism that could explain thromboembolic events occurring with DNA-based but not with RNA-based vaccines. We show that DNA-encoded mRNA coding for Spike protein can be spliced in a way that the transmembrane anchor of Spike is lost, so that nearly full-length Spike is secreted from cells. Secreted Spike variants could potentially initiate severe side effects when binding to cells via the ACE2 receptor. Avoiding such splicing events should become part of a rational vaccine design to increase safety of prospective vaccines.

Gene Therapy "Made in Germany": A Historical Perspective, Analysis of the Status Quo, and Recommendations for Action by the German Society for Gene Therapy.

Gene therapies have been successfully applied to treat severe inherited and acquired disorders. Although research and development are sufficiently well funded in Germany and while the output of scientific publications and patents is comparable with the leading nations in gene therapy, the country lags noticeably behind with regard to the number of both clinical studies and commercialized gene therapy products. In this article, we give a historical perspective on the development of gene therapy in Germany, analyze the current situation from the standpoint of the German Society for Gene Therapy (DG-GT), and define recommendations for action that would enable our country to generate biomedical and economic advantages from innovations in this sector, instead of merely importing advanced therapy medicinal products. Inter alia, we propose (1) to harmonize and simplify regulatory licensing processes to enable faster access to advanced therapies, and (2) to establish novel coordination, support and funding structures that facilitate networking of the key players. Such a center would provide the necessary infrastructure and know-how to translate cell and gene therapies to patients on the one hand, and pave the way for commercialization of these promising and innovative technologies on the other. Hence, these courses of action would not only benefit the German biotech and pharma landscape but also the society and the patients in need of new treatment options.

Insights in ChAdOx1 nCoV-19 vaccine-induced immune thrombotic thrombocytopenia.

SARS-CoV-2 vaccine ChAdOx1 nCoV-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models, and analysis of VITT patient samples, we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the ethylenediaminetetraacetic acid (EDTA)-containing vaccine. Injected vaccine increased vascular leakage in mice, leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B-cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release neutrophil extracellular traps (NETs) in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drives thrombosis in VITT. The data support a 2-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.

Persistent JunB activation in fibroblasts disrupts stem cell niche interactions enforcing skin aging.

Fibroblasts residing in the connective tissues constitute the stem cell niche, particularly in organs such as skin. Although the effect of fibroblasts on stem cell niches and organ aging is an emerging concept, the underlying mechanisms are largely unresolved. We report a mechanism of redox-dependent activation of transcription factor JunB, which, through concomitant upregulation of p16INK4A and repression of insulin growth factor-1 (IGF-1), initiates the installment of fibroblast senescence. Fibroblast senescence profoundly disrupts the metabolic and structural niche, and its essential interactions with different stem cells thus enforces depletion of stem cells pools and skin tissue decline. In fact, silencing of JunB in a fibroblast-niche-specific manner-by reinstatement of IGF-1 and p16 levels-restores skin stem cell pools and overall skin tissue integrity. Here, we report a role of JunB in the control of connective tissue niche and identified targets to combat skin aging and associated pathologies.

Virotherapy in Germany-Recent Activities in Virus Engineering, Preclinical Development, and Clinical Studies.

Virotherapy research involves the development, exploration, and application of oncolytic viruses that combine direct killing of cancer cells by viral infection, replication, and spread (oncolysis) with indirect killing by induction of anti-tumor immune responses. Oncolytic viruses can also be engineered to genetically deliver therapeutic proteins for direct or indirect cancer cell killing. In this review-as part of the special edition on "State-of-the-Art Viral Vector Gene Therapy in Germany"-the German community of virotherapists provides an overview of their recent research activities that cover endeavors from screening and engineering viruses as oncolytic cancer therapeutics to their clinical translation in investigator-initiated and sponsored multi-center trials. Preclinical research explores multiple viral platforms, including new isolates, serotypes, or fitness mutants, and pursues unique approaches to engineer them towards increased safety, shielded or targeted delivery, selective or enhanced replication, improved immune activation, delivery of therapeutic proteins or RNA, and redirecting antiviral immunity for cancer cell killing. Moreover, several oncolytic virus-based combination therapies are under investigation. Clinical trials in Germany explore the safety and potency of virotherapeutics based on parvo-, vaccinia, herpes, measles, reo-, adeno-, vesicular stomatitis, and coxsackie viruses, including viruses encoding therapeutic proteins or combinations with immune checkpoint inhibitors. These research advances represent exciting vantage points for future endeavors of the German virotherapy community collectively aimed at the implementation of effective virotherapeutics in clinical oncology.

HAP40 protein levels are huntingtin-dependent and decrease in Huntington disease.

The huntingtin-associated protein 40 (HAP40) is an abundant interactor of huntingtin (HTT). In complexes of these proteins, HAP40 tightly binds to HTT in a cleft formed by two larger domains rich in HEAT repeats, and a smaller bridge domain connecting the two. We show that HAP40 steady-state protein levels are directly dependent on HTT (both normal and mutant HTT) and that HAP40 is strongly stabilized by the interaction with HTT resulting in an at least 5-fold increase in HAP40's half-life when bound to HTT. Cellular HAP40 protein levels were reduced in primary fibroblasts and lymphoblasts of Huntington Disease (HD) patients and in brain tissue of a full-length HTT mouse model of HD, concomitant with decreased soluble HTT levels in these cell types. This data and our previous demonstration of coevolution between HTT and HAP40 and evolutionary conservation of their interaction suggest that HAP40 is an obligate interaction partner of HTT. Our observation of reduced HAP40 levels in HD invites further studies, whether HAP40 loss-of-function contributes to the pathophysiology of HD.

Transduction Enhancers Enable Efficient Human Adenovirus Type 5-Mediated Gene Transfer into Human Multipotent Mesenchymal Stromal Cells.

Human multipotent mesenchymal stromal cells (hMSCs) are currently developed as cell therapeutics for different applications, including regenerative medicine, immune modulation, and cancer treatment. The biological properties of hMSCs can be further modulated by genetic engineering. Viral vectors based on human adenovirus type 5 (HAdV-5) belong to the most frequently used vector types for genetic modification of human cells in vitro and in vivo. However, due to a lack of the primary attachment receptor coxsackievirus and adenovirus receptor (CAR) in hMSCs, HAdV-5 vectors are currently not suitable for transduction of this cell type without capsid modification. Here we present several transduction enhancers that strongly enhance HAdV-5-mediated gene transfer into both bone marrow- and adipose tissue-derived hMSCs. Polybrene, poly-l-lysine, human lactoferrin, human blood coagulation factor X, spermine, and spermidine enabled high eGFP expression levels in hMSCs. Importantly, hMSCs treated with enhancers were not affected in their migration behavior, which is a key requisite for many therapeutic applications. Exemplary, strongly increased expression of tumor necrosis factor (TNF)-stimulated gene 6 (TSG-6) (a secreted model therapeutic protein) was achieved by enhancer-facilitated HAdV-5 transduction. Thus, enhancer-mediated HAdV-5 vector transduction is a valuable method for the engineering of hMSCs, which can be further exploited for the development of innovative hMSC therapeutics.

A meta-analysis of transcriptomic profiles of Huntington's disease patients.

Description of robust transcriptomic alterations in Huntington's disease is essential to identify targets for biochemical studies and drug development. We analysed publicly available transcriptome data from the brain and blood of 220 HD patients and 241 healthy controls and identified 737 and 661 genes with robustly altered mRNA levels in the brain and blood of HD patients, respectively. In the brain, a subnetwork of 320 genes strongly correlated with HD and was enriched in transport-related genes. Bioinformatical analysis of this subnetwork highlighted CDC42, PAK1, YWHAH, NFY, DLX1, HMGN3, and PRMT3. Moreover, we found that CREB1 can regulate 78.0% of genes whose mRNA levels correlated with HD in the blood of patients. Alterations in protein transport, metabolism, transcriptional regulation, and CDC42-mediated functions are likely central features of HD. Further our data substantiate the role of transcriptional regulators that have not been reported in the context of HD (e.g. DLX1, HMGN3 and PRMT3) and strongly suggest dysregulation of NFY and its target genes across tissues. A large proportion of the identified genes such as CDC42 were also altered in Parkinson's (PD) and Alzheimer's disease (AD). The observed dysregulation of CDC42 and YWHAH in samples from HD, AD and PD patients indicates that those genes and their upstream regulators may be interesting therapeutic targets.

Pathological polyQ expansion does not alter the conformation of the Huntingtin-HAP40 complex.

The abnormal amplification of a CAG repeat in the gene coding for huntingtin (HTT) leads to Huntington's disease (HD). At the protein level, this translates into the expansion of a polyglutamine (polyQ) stretch located at the HTT N terminus, which renders HTT aggregation prone by unknown mechanisms. Here we investigated the effects of polyQ expansion on HTT in a complex with its stabilizing interaction partner huntingtin-associated protein 40 (HAP40). Surprisingly, our comprehensive biophysical, crosslinking mass spectrometry and cryo-EM experiments revealed no major differences in the conformation of HTT-HAP40 complexes of various polyQ length, including 17QHTT-HAP40 (wild type), 46QHTT-HAP40 (typical polyQ length in HD patients), and 128QHTT-HAP40 (extreme polyQ length). Thus, HTT polyQ expansion does not alter the global conformation of HTT when associated with HAP40.

EGFR-Binding Peptides: From Computational Design towards Tumor-Targeting of Adeno-Associated Virus Capsids.

The epidermal growth factor receptor (EGFR) plays a central role in the progression of many solid tumors. We used this validated target to analyze the de novo design of EGFR-binding peptides and their application for the delivery of complex payloads via rational design of a viral vector. Peptides were computationally designed to interact with the EGFR dimerization interface. Two new peptides and a reference (EDA peptide) were chemically synthesized, and their binding ability characterized. Presentation of these peptides in each of the 60 capsid proteins of recombinant adeno-associated viruses (rAAV) via a genetic based loop insertion enabled targeting of EGFR overexpressing tumor cell lines. Furthermore, tissue distribution and tumor xenograft specificity were analyzed with systemic injection in chicken egg chorioallantoic membrane (CAM) assays. Complex correlations between the targeting of the synthetic peptides and the viral vectors to cells and in ovo were observed. Overall, these data demonstrate the potential of computational design in combination with rational capsid modification for viral vector targeting opening new avenues for viral vector delivery and specifically suicide gene therapy.

The evolution of the huntingtin-associated protein 40 (HAP40) in conjunction with huntingtin.

BACKGROUND: The huntingtin-associated protein 40 (HAP40) abundantly interacts with huntingtin (HTT), the protein that is altered in Huntington's disease (HD). Therefore, we analysed the evolution of HAP40 and its interaction with HTT. RESULTS: We found that in amniotes HAP40 is encoded by a single-exon gene, whereas in all other organisms it is expressed from multi-exon genes. HAP40 co-occurs with HTT in unikonts, including filastereans such as Capsaspora owczarzaki and the amoebozoan Dictyostelium discoideum, but both proteins are absent from fungi. Outside unikonts, a few species, such as the free-living amoeboflagellate Naegleria gruberi, contain putative HTT and HAP40 orthologs. Biochemically we show that the interaction between HTT and HAP40 extends to fish, and bioinformatic analyses provide evidence for evolutionary conservation of this interaction. The closest homologue of HAP40 in current protein databases is the family of soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs). CONCLUSION: Our results indicate that the transition from a multi-exon to a single-exon gene appears to have taken place by retroposition during the divergence of amphibians and amniotes, followed by the loss of the parental multi-exon gene. Furthermore, it appears that the two proteins probably originated at the root of eukaryotes. Conservation of the interaction between HAP40 and HTT and their likely coevolution strongly indicate functional importance of this interaction.