Oleg Tolmachov

Nuclear-targeted minicircle to enhance gene transfer with non-viral vectors in vitro and in vivo

To develop more efficient non‐viral vectors, we have previously described a novel approach to attach a nuclear localisation signal (NLS) to plasmid DNA, by generating a fusion protein between the tetracycline repressor protein TetR and an SV40 NLS peptide (TetR‐NLS). The high affinity of TetR for the DNA sequence tetO is used to bind the NLS to DNA. We have now investigated the ability of this system displaying the SV40 NLS or HIV‐1 TAT peptide to enhance nuclear import of a minimised DNA construct more suitable for in vivo gene delivery: a minicircle.

Functional expression of Rab escort protein 1 following AAV2-mediated gene delivery in the retina of choroideremia mice and human cells ex vivo

Choroideremia (CHM) is an X-linked retinal degeneration of photoreceptors, the retinal pigment epithelium (RPE) and choroid caused by loss of function mutations in the CHM/REP1 gene that encodes Rab escort protein 1. As a slowly progressing monogenic retinal degeneration with a clearly identifiable phenotype and a reliable diagnosis, CHM is an ideal candidate for gene therapy. We developed a serotype 2 adeno-associated viral vector AAV2/2-CBA-REP1, which expresses REP1 under control of CMV-enhanced chicken β-actin promoter (CBA) augmented by a Woodchuck hepatitis virus post-transcriptional regulatory element. We show that the AAV2/2-CBA-REP1 vector provides strong and functional transgene expression in the D17 dog osteosarcoma cell line, CHM patient fibroblasts and CHM mouse RPE cells in vitro and in vivo. The ability to transduce human photoreceptors highly effectively with this expression cassette was confirmed in AAV2/2-CBA-GFP transduced human retinal explants ex vivo. Electroretinogram (ERG) analysis of AAV2/2-CBA-REP1 and AAV2/2-CBA-GFP-injected wild-type mouse eyes did not show toxic effects resulting from REP1 overexpression. Subretinal injections of AAV2/2-CBA-REP1 into CHM mouse retinas led to a significant increase in a- and b-wave of ERG responses in comparison to sham-injected eyes confirming that AAV2/2-CBA-REP1 is a promising vector suitable for choroideremia gene therapy in human clinical trials.

Development of a Self-assembling Nuclear Targeting Vector System Based on the Tetracycline Repressor Protein

The ultimate destination for most gene therapy vectors is the nucleus and nuclear import of potentially therapeutic DNA is one of the major barriers for nonviral vectors. We have developed a novel approach of attaching a nuclear localization sequence (NLS) peptide to DNA in a non-essential position, by generating a fusion between the tetracycline repressor protein TetR and the SV40-derived NLS peptide. The high affinity and specificity of TetR for the short DNA sequence tetO was used in these studies to bind the NLS to DNA as demonstrated by the reduced electrophoretic mobility of the TetR·tetO-DNA complexes. The protein TetR-NLS, but not control protein TetR, specifically enhances gene expression from lipofected tetO-containing DNA between 4- and 16-fold. The specific enhancement is observed in a variety of cell types, including primary and growth-arrested cells. Intracellular trafficking studies demonstrate an increased accumulation of fluorescence labeled DNA in the nucleus after TetR-NLS binding. In comparison, binding studies using the similar fusion of peptide nucleic acid (PNA) with NLS peptide, demonstrate specific binding of PNA to plasmid DNA. However, although we observed a 2–8.5-fold increase in plasmid-mediated luciferase activity with bis-PNA-NLS, control bis-PNA without an NLS sequence gave a similar increase, suggesting that the effect may not be because of a specific bis-PNA-NLS-mediated enhancement of nuclear transfer of the plasmid. Overall, we found TetRNLS-enhanced plasmid-mediated transgene expression at a similar level to that by bis-PNA-NLS or bis-PNA alone but specific to nuclear uptake and significantly more reliable and reproducible.

CHM/REP1 cDNA delivery by lentiviral vectors provides functional expression of the transgene in the retinal pigment epithelium of choroideremia mice

Choroideremia (CHM) is a progressive X‐linked degeneration of three ocular layers: photoreceptors, retinal pigment epithelium (RPE) and choroid, caused by the loss of Rab Escort Protein‐1 (REP1). As a recessive monogenic disorder, CHM is potentially curable by gene addition therapy. The present study aimed to evaluate the potential use of lentiviral vectors carrying CHM/REP1 cDNA transgene for CHM treatment.

Designing plasmid vectors.

Nonviral gene therapy vectors are commonly based on recombinant bacterial plasmids or their derivatives. The plasmids are propagated in bacteria, so, in addition to their therapeutic cargo, they necessarily contain a bacterial replication origin and a selection marker, usually a gene conferring antibiotic resistance. Structural and maintenance plasmid stability in bacteria is required for the plasmid DNA production and can be achieved by carefully choosing a combination of the therapeutic DNA sequences, replication origin, selection marker, and bacterial strain. The use of appropriate promoters, other regulatory elements, and mammalian maintenance devices ensures that the therapeutic gene or genes are adequately expressed in target human cells. Optimal immune response to the plasmid vectors can be modulated via inclusion or exclusion of DNA sequences containing immunostimulatory CpG sequence motifs. DNA fragments facilitating construction of plasmid vectors should also be considered for inclusion in the design of plasmid vectors. Techniques relying on site-specific or homologous recombination are preferred for construction of large plasmids (>15 kb), while digestion of DNA by restriction enzymes with subsequent ligation of the resulting DNA fragments continues to be the mainstream approach for generation of small- and medium-size plasmids. Rapid selection of a desired recombinant plasmid against a background of other plasmids continues to be a challenge. In this chapter, the emphasis is placed on efficient and flexible versions of DNA cloning protocols using selection of recombinant plasmids by restriction endonucleases directly in the ligation mixture.

Hhex and Cer1 Mediate the Sox17 Pathway for Cardiac Mesoderm Formation in Embryonic Stem Cells

Cardiac muscle differentiation in vivo is guided by sequential growth factor signals, including endoderm‐derived diffusible factors, impinging on cardiogenic genes in the developing mesoderm. Previously, by RNA interference in AB2.2 mouse embryonic stem cells (mESCs), we identified the endodermal transcription factor Sox17 as essential for Mesp1 induction in primitive mesoderm and subsequent cardiac muscle differentiation. However, downstream effectors of Sox17 remained to be proven functionally. In this study, we used genome‐wide profiling of Sox17‐dependent genes in AB2.2 cells, RNA interference, chromatin immunoprecipitation, and luciferase reporter genes to dissect this pathway. Sox17 was required not only for Hhex (a second endodermal transcription factor) but also for Cer1, a growth factor inhibitor from endoderm that, like Hhex, controls mesoderm patterning in Xenopus toward a cardiac fate. Suppressing Hhex or Cer1 blocked cardiac myogenesis, although at a later stage than induction of Mesp1/2. Hhex was required but not sufficient for Cer1 expression. Over‐expression of Sox17 induced endogenous Cer1 and sequence‐specific transcription of a Cer1 reporter gene. Forced expression of Cer1 was sufficient to rescue cardiac differentiation in Hhex‐deficient cells. Thus, Hhex and Cer1 are indispensable components of the Sox17 pathway for cardiopoiesis in mESCs, acting at a stage downstream from Mesp1/2. Stem Cells 2014;32:1515–1526

Designing lentiviral gene vectors

Gene therapy relies on the delivery of therapeutic genes into patients’ cells. The microdevices used to reach the cells and to transfer the gene payload are called gene vectors. Viral packaging machinery is often utilized to generate the particles transporting the cargo genes. Lentiviruses, a subgroup of retroviruses, are highly suitable for remodeling into gene transfer vectors because they offer the stability of transgene expression, the ability to reach the nuclei of the therapeutically important non-dividing cells and are known to have a low immunogenic profile. Well studied members of the lentiviruses include human immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2), feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV). It is important not to confuse “gene delivery vectors” and “gene cloning vectors”. While the former are microparticles delivering genes, the latter are replicating vehicles for the amplification of nucleic acid sequences. “Gene delivery vectors” and “gene cloning vectors” coincide when the naked DNA of replicating bacterial plasmids or replication competent viruses is used for gene delivery into cells. Viral gene delivery vectors are normally nonreplicating and should correctly be referred to as “viral vectors”, not “viruses”. Particles of viral vectors can be referred to as “virions” or “transducing particles”, because viral gene transfer is traditionally described as “transduction”. Replication deficient viral gene vector particles are similar to “defective interfering particles”, that is, faulty non-self-viable virions arising during natural viral infections and competing with non-defective virions, which were described in virology literature many years ago. Native lentiviral envelope proteins, which determine the cell range of viral infectivity (tropism) and mediate the fusion of viral and cellular membranes, are always composed from two non-covalently attached subunits, one of which (e.g. gp41 glycoprotein in HIV-1) is membrane-embedded and the other is an external subunit (e.g. gp120 glycoprotein in HIV-1). This arrangement makes lentiviruses notoriously unstable because of their tendency to shed the external subunit of the envelope protein. As the virion’s stability is a pre-requisite for the effective purification and concentration of viral vector preparations, in

Design and Production of mRNA-based Gene Vectors for Therapeutic Reprogramming of Cell Fate

Recent advances in therapeutically important cell fate reprogramming fuelled a renaissance in the use of mRNAbased gene vectors. Thus, mRNA vectors were successfully employed to induce lasting epigenetic changes in various target cells making them short-listed vector candidates for the manufacture of therapeutic engraftment materials for autologous transplantation, artificial human tissues for drug discovery via high-throughput screening projects and also for therapeutic cell trans-differentiation directly in the human body. De-differentiation of cells into ‘induced pluripotent stem cells’, transgene-directed differentiation and trans-differentiation require the simultaneous delivery of a number of regulatory factors, and, favourably, potent reprogramming vector cocktails can be straightforwardly assembled from a selection of mRNA species. In addition, several proteins can be conveniently expressed from a single mRNA using internal ribosome entry sites (IRESes) or, alternatively, fusion proteins supplemented with ‘polypeptide-cleaving’ ribosome skipping sequences. This review is focused on the design and production of cell-fate changing mRNAs.

Silencing of Transgene Expression: A Gene Therapy Perspective

The treatment of a number of diseases can be achieved through gene addition therapy, where curative transgenes are established within the patient’s cells after delivery with viral or non-viral vectors. The defective cells requiring treatment are typically differentiated; these cells or their progenitors can be targeted for therapeutic gene transfer. However, as the abundance of progenitor cells varies between different tissues and in the same tissue during the fetal, neonatal and adult stages of development, the scarcity of a particular progenitor cell pool, the paucity of spontaneous departures of progenitor cells down differentiation pathways and unclear differentiation induction conditions can complicate genetic therapeu‐ tic intervention via these cells. Nevertheless, gene transfer to progenitor cells can be a pre‐ ferred option when differentiated cells are either poorly accessible for the vector or, once differentiated, are defective beyond repair by gene therapy. Genetic conditions with consid‐ erable value in therapeutic gene transfer to progenitor cells include cystic fibrosis (CF) and severe combined immunodeficiency (SCID).

Methods of Transfection with Messenger RNA Gene Vectors

Non-viral gene delivery vectors with messenger RNA (mRNA) as a carrier of genetic information are among the staple gene transfer vectors for research in gene therapy, gene vaccination and cell fate reprogramming. As no passage of genetic cargo in and out of the nucleus is required, mRNA-based vectors typically offer the following five advantages: 1) fast start of transgene expression; 2) ability to express genes in nondividing cells with an intact nuclear envelope; 3) insensitivity to the major gene silencing mechanisms, which operate in the nucleus; 4) absence of potentially mutagenic genomic insertions; 5) high cell survival rate after transfection procedures, which do not need to disturb nuclear envelope. In addition, mRNA-based vectors offer a simple combination of various transgenes through mixing of several mRNAs in a single multi-gene cocktail or expression of a number of proteins from a single mRNA molecule using internal ribosome entry sites (IRESes), ribosome skipping sequences and proteolytic signals. However, on the downside, uncontrolled extrac‐ ellular and intracellular decay of mRNA can be a substantial hurdle for mRNAmediated gene transfer. Procedures for mRNA delivery are analogous to DNA transfer methods, which are well-established. In general, there are three actors in the gene delivery play, namely, the vector, the cell and the transfer environment. The desired outcome, that is, the efficient delivery of a gene to a target cell population, depends on the efficient interaction of all three parties. Thus, the vector should be customised for the target cell population and presented in a form that is resistant to the aggressive factors in the delivery milieu. At the same time, the delivery environ‐ ment should be adjusted to be more vector-friendly and more cell-friendly. The recipient cells should be subjected to a specific regimen or artificially modified to become receptive to gene transfer with a particular vector and resistant to the environment. As a rule, barriers outside tissues (e.g. mucus) and an aggressive