Oscar E. Simonson

Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo

While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential.

Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment

Lack of robust methods for establishment and expansion of pluripotent human embryonic stem (hES) cells still hampers development of cell therapy. Laminins (LN) are a family of highly cell-type specific basement membrane proteins important for cell adhesion, differentiation, migration and phenotype stability. Here we produce and isolate a human recombinant LN-521 isoform and develop a cell culture matrix containing LN-521 and E-cadherin, which both localize to stem cell niches in vivo. This matrix allows clonal derivation, clonal survival and long-term self-renewal of hES cells under completely chemically defined and xeno-free conditions without ROCK inhibitors. Neither LN-521 nor E-cadherin alone enable clonal survival of hES cells. The LN-521/E-cadherin matrix allows hES cell line derivation from blastocyst inner cell mass and single blastomere cells without a need to destroy the embryo. This method can facilitate the generation of hES cell lines for development of different cell types for regenerative medicine purposes.

A peptide-based vector for efficient gene transfer in vitro and in vivo.

Finding suitable nonviral delivery vehicles for nucleic acid-based therapeutics is a landmark goal in gene therapy. Cell-penetrating peptides (CPPs) are one class of delivery vectors that has been exploited for this purpose. However, since CPPs use endocytosis to enter cells, a large fraction of peptides remain trapped in endosomes. We have previously reported that stearylation of amphipathic CPPs, such as transportan 10 (TP10), dramatically increases transfection of oligonucleotides in vitro partially by promoting endosomal escape. Therefore, we aimed to evaluate whether stearyl-TP10 could be used for the delivery of plasmids as well. Our results demonstrate that stearyl-TP10 forms stable nanoparticles with plasmids that efficiently enter different cell-types in a ubiquitous manner, including primary cells, resulting in significantly higher gene expression levels than when using stearyl-Arg9 or unmodified CPPs. In fact, the transfection efficacy of stearyl-TP10 almost reached the levels of Lipofectamine 2000 (LF2000), however, without any of the observed lipofection-associated toxicities. Most importantly, stearyl-TP10/plasmid nanoparticles are nonimmunogenic, mediate efficient gene delivery in vivo, when administrated intramuscularly (i.m.) or intradermally (i.d.) without any associated toxicity in mice.Finding suitable nonviral delivery vehicles for nucleic acid-based therapeutics is a landmark goal in gene therapy. Cell-penetrating peptides (CPPs) are one class of delivery vectors that has been exploited for this purpose. However, since CPPs use endocytosis to enter cells, a large fraction of peptides remain trapped in endosomes. We have previously reported that stearylation of amphipathic CPPs, such as transportan 10 (TP10), dramatically increases transfection of oligonucleotides in vitro partially by promoting endosomal escape. Therefore, we aimed to evaluate whether stearyl-TP10 could be used for the delivery of plasmids as well. Our results demonstrate that stearyl-TP10 forms stable nanoparticles with plasmids that efficiently enter different cell-types in a ubiquitous manner, including primary cells, resulting in significantly higher gene expression levels than when using stearyl-Arg9 or unmodified CPPs. In fact, the transfection efficacy of stearyl-TP10 almost reached the levels of Lipofectamine 2000 (LF2000), however, without any of the observed lipofection-associated toxicities. Most importantly, stearyl-TP10/plasmid nanoparticles are nonimmunogenic, mediate efficient gene delivery in vivo, when administrated intramuscularly (i.m.) or intradermally (i.d.) without any associated toxicity in mice.

The safety of human pluripotent stem cells in clinical treatment.

Abstract Human pluripotent stem cells (hPSCs) have practically unlimited proliferation potential and a capability to differentiate into any cell type in the human body. Since the first derivation in 1998, they have been an attractive source of cells for regenerative medicine. Numerous ethical, technological, and regulatory complications have been hampering hPSC use in clinical applications. Human embryonic stem cells (ESCs), parthenogenetic human ESCs, human nuclear transfer ESCs, and induced pluripotent stem cells are four types of hPSCs that are different in many clinically relevant features such as propensity to epigenetic abnormalities, generation methods, and ability for development of autologous cell lines. Propensity to genetic mutations and tumorigenicity are common features of all pluripotent cells that complicate hPSC-based therapies. Several recent advances in methods of derivation, culturing, and monitoring of hPSCs have addressed many ethical concerns and technological challenges in development of clinical-grade hPSC lines. Generation of banks of such lines may be useful to minimize immune rejection of hPSC-derived allografts. In this review, we discuss different sources of hPSCs available at the moment, various safety risks associated with them, and possible solutions for successful use of hPSCs in the clinic. We also discuss ongoing clinical trials of hPSC-based treatments.

In Vivo Effects of Mesenchymal Stromal Cells in Two Patients With Severe Acute Respiratory Distress Syndrome

Mesenchymal stromal cells (MSCs) have been investigated as a treatment for various inflammatory diseases because of their immunomodulatory and reparative properties. However, many basic questions concerning their mechanisms of action after systemic infusion remain unanswered. We performed a detailed analysis of the immunomodulatory properties and proteomic profile of MSCs systemically administered to two patients with severe refractory acute respiratory distress syndrome (ARDS) on a compassionate use basis and attempted to correlate these with in vivo anti‐inflammatory actions. Both patients received 2 × 106 cells per kilogram, and each subsequently improved with resolution of respiratory, hemodynamic, and multiorgan failure. In parallel, a decrease was seen in multiple pulmonary and systemic markers of inflammation, including epithelial apoptosis, alveolar‐capillary fluid leakage, and proinflammatory cytokines, microRNAs, and chemokines. In vitro studies of the MSCs demonstrated a broad anti‐inflammatory capacity, including suppression of T‐cell responses and induction of regulatory phenotypes in T cells, monocytes, and neutrophils. Some of these in vitro potency assessments correlated with, and were relevant to, the observed in vivo actions. These experiences highlight both the mechanistic information that can be gained from clinical experience and the value of correlating in vitro potency assessments with clinical effects. The findings also suggest, but do not prove, a beneficial effect of lung protective strategies using adoptively transferred MSCs in ARDS. Appropriate randomized clinical trials are required to further assess any potential clinical efficacy and investigate the effects on in vivo inflammation.

Gene Transfer to Mouse Heart and Skeletal Muscles Using a Minicircle Expressing Human Vascular Endothelial Growth Factor

Background: Gene transfer to heart muscle is a promising modality to treat ischemic heart disease. However, current vectors are inefficient and need to be improved. A novel vector system that shows great promise is the minicircle (MC) vector being smaller than conventional plasmid vectors and devoid of bacterial sequences. Aims: To study gene transfer of MC DNA, expressing the human vascular endothelial growth factor (hVEGF), to mouse heart and skeletal muscles and to compare it with one of the efficient plasmids used in cardiovascular trials, the phVEGF165 containing the same expression cassette as the MC. Results: The MC and the phVEGF165 plasmid show similar expression patterns both in vitro and in mouse heart and skeletal muscle studies in vivo on molar basis (equal expression in heart 24 hours, 0.9 fold lower expression from MC in heart and 1.9 fold higher in skeletal muscle at 7 days), whereas on weight basis the MC construct was more efficient in skeletal muscle (5.6 fold higher expression, P < 0.05), and at least as efficient in heart (1.6 fold higher expression). Conclusions: The gene expression is similar in the 2 vector systems, so the smaller size and the fact that the MC construct is devoid of bacterial sequences and antibiotics resistance gene make the MC vector an attractive alternative for nonviral gene therapy.

Nanotechnology approaches for gene transfer

In both basic research as well as experimental gene therapy the need to transfer genetic material into a cell is of vital importance. The cellular compartment, which is the target for the genetic material, depends upon application. An siRNA that mediates silencing is preferably delivered to the cytosol while a transgene would need to end up in the nucleus for successful transcription to occur. Furthermore the ability to regulate gene expression has grown substantially since the discovery of RNA interference. In such diverse fields as medical research and agricultural pest control, the capability to alter the genetic output has been a useful tool for pushing the scientific frontiers. This review is focused on nanotechnological approaches to assemble optimised structures of nucleic acid derivatives to facilitate gene delivery as well as promoting down regulation of endogenous genes.

Bioplex Technology: Novel Synthetic Gene Delivery Pharmaceutical Based on Peptides Anchored to Nucleic Acids

Non-viral gene delivery is an important approach in order to establish safe in vivo gene therapy in the clinic. Although viral vectors currently exhibit superior gene transfer efficacy, the safety aspect of viral gene delivery is a concern. In order to improve non-viral in vivo gene delivery we have designed a pharmaceutical platform called Bioplex (biological complex). The concept of Bioplex is to link functional entities via hybridising anchors, such as Peptide Nucleic Acids (PNA), directly to naked DNA. In order to promote delivery functional entities consisting of biologically active peptides or carbohydrates, are linked to the PNA anchor. The PNA acts as genetic glue and hybridises with DNA in a sequence specific manner. By using functional entities, which elicit receptor-mediated endocytosis, improved endosomal escape and enhance nuclear entry we wish to improve the transfer of genetic material into the cell. An important aspect is that the functional entities should also have tissue-targeting properties in vivo. Examples of functional entities investigated to date are the Simian virus 40 nuclear localisation signal to improve nuclear uptake and different carbohydrate ligands in order to achieve receptor specific uptake. The delivery system is also endowed with regulatory capability, since the release of functional entities can be controlled. The aim is to create a safe, pharmaceutically defined and stable delivery system for nucleic acids with enhanced transfection properties that can be used in the clinic.

Sequence‐specific inhibition of RNA polymerase III‐dependent transcription using Zorro locked nucleic acid (LNA)

RNA polymerase III (pol III)‐dependent transcripts are involved in many fundamental activities in a cell, such as splicing and protein synthesis. They also regulate cell growth and influence tumor formation. During recent years vector‐based systems for expression of short hairpin (sh) RNA under the control of a pol III promoter have been developed as gene‐based medicines. Therefore, there is an increasing interest in means to regulate pol III‐dependent transcription. Recently, we have developed a novel anti‐gene molecule ‘Zorro LNA (Locked Nucleic Acid)’, which simultaneously hybridizes to both strands of super‐coiled DNA and potently inhibits RNA polymerase II‐derived transcription. We have now applied Zorro LNA in an attempt to also control U6 promoter‐driven expression of shRNA.

Fatty acid-spermine conjugates as DNA carriers for nonviral in vivo gene delivery

The lack of efficient in vivo gene delivery is a well-known shortcoming of nonviral delivery vectors, in particular of chemical vectors. We developed a series of novel nonviral carriers for plasmid-based in vivo gene delivery. This new transport device is based on the assembly of DNA plasmids with synthetic derivatives of naturally occurring molecules—fatty acid–spermine conjugates (or lipospermines). We tested the ability of these fatty acid conjugates to interact with plasmid DNA (pDNA) and found that they formed DNA nanocomplexes, which are protected from DNase I degradation. This protection was shown to directly correlate with the length of the aliphatic component. However, this increase in the length of the hydrocarbon chain resulted in increased toxicity. The cationic lipids used for transfection typically have a C16 and C18 hydrocarbon chain. Interestingly, toxicity studies, together with further characterization studies, suggested that the two most suitable candidates for in vivo delivery are those with the shortest hydrocarbon chain, butanoyl- and decanoylspermine. Morphological characterization of DNA nanocomplexes resulting from these lipospermines showed the formation of a homogenous population, with the diameter ranging approximately from 40 to 200 nm. Butanoylspermine was found to be the most promising carrier from this series, resulting in a significantly increased gene expression, in relation to naked plasmid, in both tissues herein targeted (dermis and M. tibialis anterior). Thus, we established a correlation between the in vitro properties of the ensuing DNA nanocarriers and their efficient in vivo gene expression.