CRISISS: A Novel, Transcriptionally and Post-Translationally Inducible CRISPR/Cas9-Based Cellular Suicide Switch.

With the ever-increasing developing rate of gene and cellular therapy applications and growing accessibility due to products receiving regulatory approval, the need for effective and reliable safety mechanisms to prevent or eliminate potentially fatal side effects is of the utmost importance. In this study, we present the CRISPR-induced suicide switch (CRISISS) as a tool to eliminate genetically modified cells in an inducible and highly efficient manner by targeting Cas9 to highly repetitive Alu retrotransposons in the human genome, causing irreparable genomic fragmentation by the Cas9 nuclease and resulting cell death. The suicide switch components, including expression cassettes for a transcriptionally and post-translationally inducible Cas9 and an Alu-specific single-guide RNA, were integrated into the genome of target cells via Sleeping-Beauty-mediated transposition. The resulting transgenic cells did not show signs of any impact on overall fitness when uninduced, as unintended background expression, background DNA damage response and background cell killing were not observed. When induced, however, a strong expression of Cas9, a strong DNA damage response and a rapid halt of cell proliferation coupled with near complete cell death within four days post-induction were seen. With this proof-of-concept study, we present a novel and promising approach for a robust suicide switch with potential utility for gene and cell therapy in the future.

Antiangiogenic Vascular Endothelial Growth Factor-Blocking Peptides Displayed on the Capsid of an Infectious Oncolytic Parvovirus: Assembly and Immune Interactions.

As many tumor cells synthetize vascular endothelial growth factors (VEGF) that promote neo-vascularization and metastasis, frontline cancer therapies often administer anti-VEGF (α-VEGF) antibodies. To target the oncolytic parvovirus minute virus of mice (MVM) to the tumor vasculature, we studied the functional tolerance, evasion of neutralization, and induction of α-VEGF antibodies of chimeric viruses in which the footprint of a neutralizing monoclonal antibody within the 3-fold capsid spike was replaced by VEGF-blocking peptides: P6L (PQPRPL) and A7R (ATWLPPR). Both peptides allowed viral genome replication and nuclear translocation of chimeric capsid subunits. MVM-P6L efficiently propagated in culture, exposing the heterologous peptide on the capsid surface, and evaded neutralization by the anti-spike monoclonal antibody. In contrast, MVM-A7R yielded low infectious titers and was poorly recognized by an α-A7R monoclonal antibody. MVM-A7R showed a deficient assembly pattern, suggesting that A7R impaired a transitional configuration that the subunits must undergo in the 3-fold axis to close up the capsid shell. The MVM-A7R chimeric virus consistently evolved in culture into a mutant carrying the P6Q amino acid substitution within the A7R sequence, which restored normal capsid assembly and infectivity. Consistent with this finding, anti-native VEGF antibodies were induced in mice by a single injection of MVM-A7R empty capsids, but not by MVM-A7R virions. This fundamental study provides insights to endow an infectious parvovirus with immune antineovascularization and evasion capacities by replacing an antibody footprint in the capsid 3-fold axis with VEGF-blocking peptides, and it also illustrates the evolutionary capacity of single-stranded DNA (ssDNA) viruses to overcome engineered capsid structural restrictions.IMPORTANCE Targeting the VEGF signaling required for neovascularization by vaccination with chimeric capsids of oncolytic viruses may boost therapy for solid tumors. VEGF-blocking peptides (VEbp) engineered in the capsid 3-fold axis endowed the infectious parvovirus MVM with the ability to induce α-VEGF antibodies without adjuvant and to evade neutralization by MVM-specific antibodies. However, these properties may be compromised by structural restraints that the capsid imposes on the peptide configuration and by misassembly caused by the heterologous peptides. Significantly, chimeric MVM-VEbp resolved the structural restrictions by selecting mutations within the engineered peptides that restored efficient capsid assembly. These data show the promise of antineovascularization vaccines using chimeric VEbp-icosahedral capsids of oncolytic viruses but also raise safety concerns regarding the genetic stability of manipulated infectious parvoviruses in cancer and gene therapies.

Efficient Non-viral Gene Delivery into Human Hematopoietic Stem Cells by Minicircle Sleeping Beauty Transposon Vectors.

The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34+ cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size. As compared to conventional plasmids, delivery of the SB transposon system as minicircle DNA is ∼20 times more efficient, and it is associated with up to a 50% reduction in cellular toxicity in human CD34+ cells. Moreover, providing the SB transposase in the form of synthetic mRNA enabled us to further increase the efficacy and biosafety of stable gene delivery into hematopoietic progenitors ex vivo. Genome-wide insertion site profiling revealed a close-to-random distribution of SB transposon integrants, which is characteristically different from gammaretroviral and lentiviral integrations in HSPCs. Transplantation of gene-marked CD34+ cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution, which was most efficient when the SB transposase was supplied as mRNA and nucleofected cells were maintained for 4-8 days in culture before transplantation. Collectively, implementation of minicircle and mRNA technologies allowed us to further refine the SB transposon system in the context of HSPC gene delivery to ultimately meet clinical demands of an efficient and safe non-viral gene therapy protocol.

Dissecting the role of epidermal growth factor receptor catalytic activity during liver regeneration and hepatocarcinogenesis.

UNLABELLED: Different data support a role for the epidermal growth factor receptor (EGFR) pathway during liver regeneration and hepatocarcinogenesis. However, important issues, such as the precise mechanisms mediating its actions and the unique versus redundant functions, have not been fully defined. Here, we present a novel transgenic mouse model expressing a hepatocyte-specific truncated form of human EGFR, which acts as negative dominant mutant (ΔEGFR) and allows definition of its tyrosine kinase-dependent functions. Results indicate a critical role for EGFR catalytic activity during the early stages of liver regeneration. Thus, after two-thirds partial hepatectomy, ΔEGFR livers displayed lower and delayed proliferation and lower activation of proliferative signals, which correlated with overactivation of the transforming growth factor-ß pathway. Altered regenerative response was associated with amplification of cytostatic effects of transforming growth factor-ß through induction of cell cycle negative regulators. Interestingly, lipid synthesis was severely inhibited in ΔEGFR livers after partial hepatectomy, revealing a new function for EGFR kinase activity as a lipid metabolism regulator in regenerating hepatocytes. In spite of these profound alterations, ΔEGFR livers were able to recover liver mass by overactivating compensatory signals, such as c-Met. Our results also indicate that EGFR catalytic activity is critical in the early preneoplastic stages of the liver because ΔEGFR mice showed a delay in the appearance of diethyl-nitrosamine-induced tumors, which correlated with decreased proliferation and delay in the diethyl-nitrosamine-induced inflammatory process. CONCLUSION: These studies demonstrate that EGFR catalytic activity is critical during the initial phases of both liver regeneration and carcinogenesis and provide key mechanistic insights into how this kinase acts to regulate liver pathophysiology. (Hepatology 2016;63:604-619).

A slender tract of glycine residues is required for translocation of the VP2 protein N-terminal domain through the parvovirus MVM capsid channel to initiate infection.

Viruses constitute paradigms to study conformational dynamics in biomacromolecular assemblies. Infection by the parvovirus MVM (minute virus of mice) requires a conformational rearrangement that involves the intracellular externalization through capsid channels of the 2Nt (N-terminal region of VP2). We have investigated the role in this process of conserved glycine residues in an extended glycine-rich tract located immediately after 2Nt. Based on the virus structure, residues with hydrophobic side chains of increasing volume were substituted for glycine residues 31 or 33. Mutations had no effect on capsid assembly or stability, but inhibited virus infectivity. All mutations, except those to alanine residues which had minor effects, impaired 2Nt externalization in nuclear maturing virions and in purified virions, to an extent that correlated with the side chain size. Different biochemical and biophysical analyses were consistent with this result. Importantly, all of the tested glycine residue replacements impaired the capacity of the virion to initiate infection, at ratios correlating with their restrictive effects on 2Nt externalization. Thus small residues within the evolutionarily conserved glycine-rich tract facilitate 2Nt externalization through the capsid channel, as required by this virus to initiate cell entry. The results demonstrate the exquisite dependence on geometric constraints of a biologically relevant translocation event in a biomolecular complex.

Intracellular virion traffic to the endosome driven by cell type specific sialic acid receptors determines parvovirus tropism.

Parvoviruses are promising anticancer and gene therapy agents, but a deep knowledge of the entry process is crucial to exploit their therapeutic potential. We addressed this issue while attempting to retarget the oncolytic parvovirus minute virus of mice (MVMp) to the tumor vasculature. Residues at three functional domains of the icosahedral capsid were substituted by rational design with peptides competing with the vascular endothelial growth factor. Most substitutions impaired virus maturation, though some yielded infectious chimeric virions, and substitutions in a dimple at the twofold axis that allocates sialic acid (SIA) receptors altered viral tropism. One dimple-modified chimeric virion was efficiently attached as MVMp to α2-linked SIA moieties, but the infection was impaired by the binding to some inhibitory α2-3,-6,-8 SIA pseudoreceptors, which hampers intracellular virus traffic to the endosome in a cell type-dependent manner. Infectious from nonproductive traffic could be mechanistically discriminated by an endosomal drastic capsid structural transition comprising the cleavage of some VP2-Nt sequences and its associated VP1-Nt exposure. Correspondingly, neuraminidase removal of inhibitory SIA moieties enhanced the infection quantitatively, correlating to the restored virus traffic to the endosome and the extent of VP2-Nt cleavage/VP1-Nt exposure. This study illustrates (i) structural constraints to retarget parvoviruses with evolutionary adopted narrow grooves allocating small SIA receptors, (ii) the possibility to enhance parvovirus oncolysis by relaxing the glycan network on the cancer cell surface, and (iii) the major role played by the attachment to cell type-specific SIAs in the intracellular virus traffic to the endosome, which may determine parvovirus tropism and host range.

RNA-guided retargeting of Sleeping Beauty transposition in human cells.

An ideal tool for gene therapy would enable efficient gene integration at predetermined sites in the human genome. Here we demonstrate biased genome-wide integration of the Sleeping Beauty (SB) transposon by combining it with components of the CRISPR/Cas9 system. We provide proof-of-concept that it is possible to influence the target site selection of SB by fusing it to a catalytically inactive Cas9 (dCas9) and by providing a single guide RNA (sgRNA) against the human Alu retrotransposon. Enrichment of transposon integrations was dependent on the sgRNA, and occurred in an asymmetric pattern with a bias towards sites in a relatively narrow, 300 bp window downstream of the sgRNA targets. Our data indicate that the targeting mechanism specified by CRISPR/Cas9 forces integration into genomic regions that are otherwise poor targets for SB transposition. Future modifications of this technology may allow the development of methods for specific gene insertion for precision genetic engineering.

A highly soluble Sleeping Beauty transposase improves control of gene insertion.

The Sleeping Beauty (SB) transposon system is an efficient non-viral gene transfer tool in mammalian cells, but its broad use has been hampered by uncontrolled transposase gene activity from DNA vectors, posing a risk of genome instability, and by the inability to use the transposase protein directly. In this study, we used rational protein design based on the crystal structure of the hyperactive SB100X variant to create an SB transposase (high-solubility SB, hsSB) with enhanced solubility and stability. We demonstrate that hsSB can be delivered with transposon DNA to genetically modify cell lines and embryonic, hematopoietic and induced pluripotent stem cells (iPSCs), overcoming uncontrolled transposase activity. We used hsSB to generate chimeric antigen receptor (CAR) T cells, which exhibit potent antitumor activity in vitro and in xenograft mice. We found that hsSB spontaneously penetrates cells, enabling modification of iPSCs and generation of CAR T cells without the use of transfection reagents. Titration of hsSB to modulate genomic integration frequency achieved as few as two integrations per genome.

One-step Multiplex Transgenesis via Sleeping Beauty Transposition in Cattle.

Genetically modified cattle are important for developing new biomedical models and for an improved understanding of the pathophysiology of zoonotic diseases. However, genome editing and genetic engineering based on somatic cell nuclear transfer suffer from a low overall efficiency. Here, we established a highly efficient one-step multiplex gene transfer system into the bovine genome.

Essential role of the unordered VP2 n-terminal domain of the parvovirus MVM capsid in nuclear assembly and endosomal enlargement of the virion fivefold channel for cell entry.

The unordered N-termini of parvovirus capsid proteins (Nt) are translocated through a channel at the icosahedral five-fold axis to serve for virus traffick. Heterologous peptides were genetically inserted at the Nt of MVM to study their functional tolerance to manipulations. Insertion of a 5T4-single-chain antibody at VP2-Nt (2Nt) yielded chimeric capsid subunits failing to enter the nucleus. The VEGFR2-binding peptide (V1) inserted at both 2Nt and VP1-Nt efficiently assembled in virions, but V1 disrupted VP1 and VP2 entry functions. The VP2 defect correlated with restricted externalization of V1-2Nt out of the coat. The specific infectivity of MVM and wtVP-pseudotyped mosaic MVM-V1 virions, upon heating and/or partial 2Nt cleavage, demonstrated that some 2Nt domains become intracellularly translocated out of the virus shell and cleaved to initiate entry. The V1 insertion defines a VP2-driven endosomal enlargement of the channel as an essential structural rearrangement performed by the MVM virion to infect.

Cell fusion reprogramming leads to a specific hepatic expression pattern during mouse bone marrow derived hepatocyte formation in vivo.

The fusion of bone marrow (BM) hematopoietic cells with hepatocytes to generate BM derived hepatocytes (BMDH) is a natural process, which is enhanced in damaged tissues. However, the reprogramming needed to generate BMDH and the identity of the resultant cells is essentially unknown. In a mouse model of chronic liver damage, here we identify a modification in the chromatin structure of the hematopoietic nucleus during BMDH formation, accompanied by the loss of the key hematopoietic transcription factor PU.1/Sfpi1 (SFFV proviral integration 1) and gain of the key hepatic transcriptional regulator HNF-1A homeobox A (HNF-1A/Hnf1a). Through genome-wide expression analysis of laser captured BMDH, a differential gene expression pattern was detected and the chromatin changes observed were confirmed at the level of chromatin regulator genes. Similarly, Tranforming Growth Factor-ß1 (TGF-ß(1)) and neurotransmitter (e.g. Prostaglandin E Receptor 4 [Ptger4]) pathway genes were over-expressed. In summary, in vivo BMDH generation is a process in which the hematopoietic cell nucleus changes its identity and acquires hepatic features. These BMDHs have their own cell identity characterized by an expression pattern different from hematopoietic cells or hepatocytes. The role of these BMDHs in the liver requires further investigation.

Functional relevance of amino acid residues involved in interactions with ordered nucleic acid in a spherical virus.

In the spherical virion of the parvovirus minute virus of mice, several amino acid side chains of the capsid were previously found to be involved in interactions with the viral single-stranded DNA molecule. We have individually truncated by mutation to alanine many (ten) of these side chains and analyzed the effects on capsid assembly, stability and conformation, viral DNA encapsidation, and virion infectivity. Mutation of residues Tyr-270, Asp-273, or Asp-474 led to a drastic reduction in infectivity. Mutant Y270A was defective in capsid assembly; mutant D273A formed stable capsids, but it was essentially unable to encapsidate the viral DNA or to externalize the N terminus of the capsid protein VP2, a connected conformational event. Mutation of residues Asp-58, Trp-60, Asn-183, Thr-267, or Lys-471 led to a moderate reduction in infectivity. None of these mutations had an effect on capsid assembly or stability, or on the DNA encapsidation process. However, those five mutant virions were substantially less stable than the parental virion in thermal inactivation assays. The results with this model spherical virus indicate that several capsid residues that are found to be involved in polar interactions or multiple hydrophobic contacts with the viral DNA molecule contribute to preserving the active conformation of the infectious viral particle. Their effect appears to be mediated by the non-covalent interactions they establish with the viral DNA. In addition, at least one acidic residue at each DNA-binding region is needed for DNA packaging.