Engineering AAV-2 Targeting Vectors: A New Insertion Site and scFv Driven Vectors

Gene therapy can be defined as the introduction of nucleic acids into cells with the purpose of altering the course of a medical condition or disease. In many clinical settings, efficient and successful gene therapy relies on the delivery of genes to specific cells in the human body. Such specific delivery can only be achieved through the design of vehicles/vectors that are able to recognize the target cell - targeting. Adeno-associated virus type 2, a non-pathogenic human virus, has received an increased amount of attention as a vector for gene therapy since it was first cloned into a bacterial plasmid in 1982. Although the first attempt to construct an AAV-2 targeting vector made use of a single chain antibody fragment, it was the insertion of small peptide ligands into the AAV-2 capsid that marked the beginning of AAV-2 vector targeting. Sequence alignment of AAV-2 with CPV led to the discovery of amino acid position 587. Several reports have shown that small peptide ligands, once inserted in this position, are able to mediate transduction of the respective target cells, and that target specificity is retained in vivo. In this work, the available three dimensional structure of AAV-2 is used for the first time for the rational design and construction of targeting vectors. In silico analysis revealed that insertions at position 453 should result in a better exposure of the inserted peptides and, as a consequence, in a more efficient interaction with the target receptor. The RGD4C peptide - a ligand for aVb5 and aVb3 integrins - was inserted in position 453 and/or 587. Moreover, loss of AAV-2 wild-type (wt) tropism was achieved by R585A and R588A - A2 - mutations that abolish binding to the primary receptor - heparan sulfate proteoglycan, HSPG. ( ) NCBI's Conserved Domain Database was used to identify 453 homologues in other vector systems. In summary, 453 emerged as a suitable position for the insertion of targeting peptides in AAV-2 and other vector systems. Double insertion mutants will have to be analyzed for each specific case. The increased targeting efficiency after single point mutations of residues linearly distant from the inserted peptide shows for the first time how such mutations can indeed be relevant for the design of targeting mutants. Moreover, high-throughput selection protocols emerge as master tools and should be put into practice for the identification of similar mutants and for the optimization of targeting vectors. The rising number of reports on the high efficiency of antibody fragments for the construction of targeting molecules motivated the fusion of single chain antibody (scFv) fragments to the capsid of AAV-2. Being most likely too large for insertions in non-terminal positions like 453 or 587, we decided to genetically fuse a scFv to the N-terminus of a viral protein - VP. Furthermore, vectors containing a GFP-VP2 fusion protein have previously been shown to be useful tools for infectious biology studies. Our analysis of this GFP labeled vectors revealed that at least a part of the GFP molecule is present on the outer surface of the capsid. These observations led to generation of an anti-CD30 scFv fused to the N-terminus of a GFP molecule that was fused to the N-terminus of VP2. Optimization of the packaging procedure, by increasing the amount of the fusion protein encoding plasmid, resulted in an efficient packaging of scFv-AAV-2 mutants. Mutants were able to effectively transduce HeLa cells and to bind an anti-idiotypic antibody homologue to the recognized antigen epitope. Despite this, targeting mutants were not able to specifically transduce CD30 positive cells. Furthermore it was not possible to detect vector DNA with target cells neither 1 nor 4 hours post-transduction. Engineering similar constructs without the GFP molecule and with or without linker sequences did not result in an improved binding or transduction efficiencies. Substitution of the anti-CD30 scFv by an anti-CEA scFv or an anti-CA19 scFv revealed that the results observed with the anti-CD30 scFV targeting mutants were not idiotype specific. When compared to studies made with whole immunoglobulins, the low vector particle concentration emerges as one of the limiting steps to the application of such targeting approaches. Moreover, while each capsid possesses sixty repeats of G453, only five VP2 proteins exist per capsid. Despite this, the scFv-GFP-VP2 fusion represents the largest fusion (~127 kDa in total) ever assembled in an AAV capsid. This, together with the fact that both GFP and anti-CD30 scFv molecules could be recognized by respective antibodies reinforces the idea that the N-terminus of VP2 can indeed be displayed on the outer surface of the capsid. Our results open the door to many other therapeutic designs where the vector can be used as a carrier for genes and at the same time for high molecular weight proteins assembled in pentavalent forms

Heterochromatin-Driven Nuclear Softening Protects the Genome against Mechanical Stress-Induced Damage

Summary Tissue homeostasis requires maintenance of functional integrity under stress. A central source of stress is mechanical force that acts on cells, their nuclei, and chromatin, but how the genome is protected against mechanical stress is unclear. We show that mechanical stretch deforms the nucleus, which cells initially counteract via a calcium-dependent nuclear softening driven by loss of H3K9me3-marked heterochromatin. The resulting changes in chromatin rheology and architecture are required to insulate genetic material from mechanical force. Failure to mount this nuclear mechanoresponse results in DNA damage. Persistent, high-amplitude stretch induces supracellular alignment of tissue to redistribute mechanical energy before it reaches the nucleus. This tissue-scale mechanoadaptation functions through a separate pathway mediated by cell-cell contacts and allows cells/tissues to switch off nuclear mechanotransduction to restore initial chromatin state. Our work identifies an unconventional role of chromatin in altering its own mechanical state to maintain genome integrity in response to deformation.Peer reviewe

Heparan Sulfate Proteoglycan Binding Properties of Adeno-Associated Virus Retargeting Mutants and Consequences for Their In Vivo Tropism

Adeno-associated virus type 2 (AAV-2) targeting vectors have been generated by insertion of ligand peptides into the viral capsid at amino acid position 587. This procedure ablates binding of heparan sulfate proteoglycan (HSPG), AAV-2's primary receptor, in some but not all mutants. Using an AAV-2 display library, we investigated molecular mechanisms responsible for this phenotype, demonstrating that peptides containing a net negative charge are prone to confer an HSPG nonbinding phenotype. Interestingly, in vivo studies correlated the inability to bind to HSPG with liver and spleen detargeting in mice after systemic application, suggesting several strategies to improve efficiency of AAV-2 retargeting to alternative tissues

ER-associated RNA silencing promotes ER quality control

The endoplasmic reticulum (ER) coordinates mRNA translation and processing of secreted and endomembrane proteins. ER-associated degradation (ERAD) prevents the accumulation of misfolded proteins in the ER, but the physiological regulation of this process remains poorly characterized. Here, in a genetic screen using an ERAD model substrate in Caenorhabditis elegans, we identified an anti-viral RNA interference pathway, referred to as ER-associated RNA silencing (ERAS), which acts together with ERAD to preserve ER homeostasis and function. Induced by ER stress, ERAS is mediated by the Argonaute protein RDE-1/AGO2, is conserved in mammals and promotes ER-associated RNA turnover. ERAS and ERAD are complementary, as simultaneous inactivation of both quality-control pathways leads to increased ER stress, reduced protein quality control and impaired intestinal integrity. Collectively, our findings indicate that ER homeostasis and organismal health are protected by synergistic functions of ERAS and ERAD

Label-Free Protein-RNA Interactome Analysis Identifies Khsrp Signaling Downstream of the p38/Mk2 Kinase Complex as a Critical Modulator of Cell Cycle Progression

<div><p>Growing evidence suggests a key role for RNA binding proteins (RBPs) in genome stability programs. Additionally, recent developments in RNA sequencing technologies, as well as mass-spectrometry techniques, have greatly expanded our knowledge on protein-RNA interactions. We here use full transcriptome sequencing and label-free LC/MS/MS to identify global changes in protein-RNA interactions in response to etoposide-induced genotoxic stress. We show that RBPs have distinct binding patterns in response to genotoxic stress and that inactivation of the RBP regulator module, p38/MK2, can affect the entire spectrum of protein-RNA interactions that take place in response to stress. In addition to validating the role of known RBPs like Srsf1, Srsf2, Elavl1 in the genotoxic stress response, we add a new collection of RBPs to the DNA damage response. We identify Khsrp as a highly regulated RBP in response to genotoxic stress and further validate its role as a driver of the G₁/S transition through the suppression of <i>Cdkn1a^P21</i> transcripts. Finally, we identify KHSRP as an indicator of overall survival, as well as disease free survival in glioblastoma multiforme.</p></div

Changes in protein-RNA interactions in response to etoposide treatment.

<p><b>(A)</b> Etoposide induces DNA double strand breaks after 6h of treatment as reported by the DSB marker <b>γ</b>-H2AX. <b>(B)</b> Schematics of the experimental procedure for the purification of RBPs show how UV-mediated crosslinking of RNA to interacting proteins was followed by poly-A selection to identify RBPs through LC/MS/MS. <b>(C)</b> Crosslinking followed by purification of mRNA-interacting proteins and nano LC/MS/MS identified 335 protein group hits of which 287 were known as RBPs. <b>(D)</b> Heat map of differentially abundant RBPs. <b>(E)</b> Vulcano plot representing changes in mRNA-protein interactions in response to etoposide treatment identifies Khsrp as the most significantly changed RBP in response to etoposide treatment. <b>(F)</b> Immunoblot analysis of proteins co-purified with poly-A-containing RNA validates protein-RNA interactome changes identified by label free LC/MS/MS (right panel). Whole cell lysates show no significant changes in protein levels of analyzed RBPs (left panel).</p

Khsrp-dependent regulation of <i>Cdkn1a</i>^<i>P21</i>.

<p><b>(A)</b> Cell cycle analysis and <b>(B)</b> mitotic index of wildtype and <i>Khsrp</i>^{<b><i>-/-</i></b>} MEFs expose a <i>Khsrp</i>-dependent <b>(A)</b> accumulation of cells in G_<b>1</b> and <b>(B)</b> decreased cycling rates. <b>(C)</b> Transcript and <b>(D)</b> protein levels of <i>Cdkn1a</i>^{<b><i>P21</i></b>} in <i>Khsrp</i>^{<b><i>-/-</i></b>} cells measured by qPCR and immunoblotting reveals an increase of Cdkn1a^{<b><i>P21</i></b>} protein levels unrelated to <i>Cdkn1a</i>^{<b><i>P21</i></b>} mRNA levels.</p

<i>KHSRP</i> transcript levels predict survival of human glioblastoma patients.

<p><b>(A)</b> Overall survival (OS) curves and <b>(B)</b> disease free survival (DFS) curves show an increased OS and DFS of patients bearing tumors with higher <i>KHSRP</i> transcript levels. <b>(C,D)</b> Although not significant, the inverted tendency can be seen when segregating patients in agreement with their tumor <i>CDKN1A</i>^{<b><i>P21</i></b>} transcript levels. Upper and lower quartiles are shown.</p

Murine embryonic fibroblasts arrest in G₂ in response to etoposide treatment.

<p><b>(A)</b> Gene expression changes identified by RNA-Seq following 6h of treatment with 20μM etoposide were analyzed for enrichments in GO terms. Cell cycle, and specifically mitotic processes, emerged in the top 10 most significant GO terms. <b>(B)</b> Protein-protein interactions-based network expansion of RBPs showing differential protein-RNA interactions upon etoposide treatment identifies enrichments for cyclin-dependent processes. <b>(C)</b> Cell cycle analysis of untreated (black line) and etoposide-treated (gray) cells reveals an accumulation of cells with 4N DNA content and decreased staining of the mitotic marker pHH3.</p

Inference of outlying RBP-client interactions from changes in client transcripts.

<p><b>(A)</b> Using gene expression levels, numbers of altered client mRNAs were plotted against number of known client mRNAs for each respective RBP. A linear correlation could be identified between the number of changed client mRNAs and known client mRNAs. <b>(B)</b> Studentized residuals (outlyingness), leverage (potential to influence the linear model) and influence analysis (represented by the size to point) are represented through influence plots. Data points perturbing the model were identified by high leverage and studentized residuals. Outliers representing RBPs with higher number of changed client mRNAs were identified through high absolute values of standardized residuals. The same was done by <b>(C)</b> plotting number of upregulated clients against number of changed clients, as well as using vector information on <b>(D)</b> differential promoter usage, <b>(E)</b> differential splicing, and <b>(F)</b> differential CDS. DNA damage-related RBPs—Elavl1, Tia1, Tial1, Srsf1, Srsf2 could be identified through RBP-client analysis.</p