Exploitation of Herpesvirus Immune Evasion Strategies to Modify the Immunogenicity of Human Mesenchymal Stem Cell Transplants

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent cells residing in the connective tissue of many organs and holding great potential for tissue repair. In culture, human MSCs (hMSCs) are capable of extensive proliferation without showing chromosomal aberrations. Large numbers of hMSCs can thus be acquired from small samples of easily obtainable tissues like fat and bone marrow. MSCs can contribute to regeneration indirectly by secretion of cytokines or directly by differentiation into specialized cell types. The latter mechanism requires their long-term acceptance by the recipient. Although MSCs do not elicit immune responses in vitro, animal studies have revealed that allogeneic and xenogeneic MSCs are rejected. METHODOLOGY/PRINCIPAL FINDINGS: We aim to overcome MSC immune rejection through permanent down-regulation of major histocompatibility complex (MHC) class I proteins on the surface of these MHC class II-negative cells through the use of viral immune evasion proteins. Transduction of hMSCs with a retroviral vector encoding the human cytomegalovirus US11 protein resulted in strong inhibition of MHC class I surface expression. When transplanted into immunocompetent mice, persistence of the US11-expressing and HLA-ABC-negative hMSCs at levels resembling those found in immunodeficient (i.e., NOD/SCID) mice could be attained provided that recipients' natural killer (NK) cells were depleted prior to cell transplantation. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate the potential utility of herpesviral immunoevasins to prevent rejection of xenogeneic MSCs. The observation that down-regulation of MHC class I surface expression renders hMSCs vulnerable to NK cell recognition and cytolysis implies that multiple viral immune evasion proteins are likely required to make hMSCs non-immunogenic and thereby universally transplantable

Anomer-Equilibrated Streptozotocin Solution for the Induction of Experimental Diabetes in Mice (Mus musculus)

Streptozotocin is widely used to induce diabetes in laboratory animals through multiple low-dose or single high-dose intraperitoneal injections. HPLC analysis has shown that the composition of the solution may change considerably during the first 2 h after dissolution due to equilibration of the 2 anomers (α and β) of streptozotocin. Because of the drug's alleged instability in solution, the typical recommendation is to administer streptozotocin within 10 min after dissolution. We compared the induction of diabetes in NOD/SCID mice by injection of a single high dose of freshly made or anomer-equilibrated streptozotocin solution. Solutions were prepared from dry compound containing 85% of the α anomer, which is the more toxic of the 2. Body weight and nonfasting blood glucose levels were measured weekly for 8 wk. Both solutions induced long-term hyperglycemia, but blood glucose levels and mortality were higher and damage to pancreatic islands more pronounced in the mice receiving freshly prepared solution. A small proportion of mice did not respond in both treatment groups. If stored at 4 °C in the dark, the anomer-equilibrated solution retains its biologic activity for at least 40 d; under those conditions the streptozotocin content decreases by 0.1% daily, as determined by HPLC. Anomer-equilibrated streptozotocin solution has several practical advantages, and we recommend its use as standard for the induction of experimental diabetes because this practice may improve reproducibility and comparison of results between different laboratories

Adenoviral Vectors Stimulate Glucagon Transcription in Human Mesenchymal Stem Cells Expressing Pancreatic Transcription Factors

<div><p>Viral gene carriers are being widely used as gene transfer systems in (trans)differentiation and reprogramming strategies. Forced expression of key regulators of pancreatic differentiation in stem cells, liver cells, pancreatic duct cells, or cells from the exocrine pancreas, can lead to the initiation of endocrine pancreatic differentiation. While several viral vector systems have been employed in such studies, the results reported with adenovirus vectors have been the most promising <em>in vitro</em> and <em>in vivo</em>. In this study, we examined whether the viral vector system itself could impact the differentiation capacity of human bone-marrow derived mesenchymal stem cells (hMSCs) toward the endocrine lineage. Lentivirus-mediated expression of Pdx-1, Ngn-3, and Maf-A alone or in combination does not lead to robust expression of any of the endocrine hormones (i.e. insulin, glucagon and somatostatin) in hMSCs. Remarkably, subsequent transduction of these genetically modified cells with an irrelevant early region 1 (E1)-deleted adenoviral vector potentiates the differentiation stimulus and promotes glucagon gene expression in hMSCs by affecting the chromatin structure. This adenovirus stimulation was observed upon infection with an E1-deleted adenovirus vector, but not after exposure to helper-dependent adenovirus vectors, pointing at the involvement of genes retained in the E1-deleted adenovirus vector in this phenomenon. Lentivirus mediated expression of the adenovirus E4-ORF3 mimics the adenovirus effect. From these data we conclude that E1-deleted adenoviral vectors are not inert gene-transfer vectors and contribute to the modulation of the cellular differentiation pathways.</p> </div

E1-deleted adenoviral vector transduction triggers epigenetic changes in BM-MSC.

<p>(A–B) LV-CMV-GFP or LV-CMV-Pdx-1-modified BM-MSC were treated for 3 days with TSA (1 µM). Pdx-1 and glucagon gene expression was assessed by RT-PCR (A) or qPCR (B). GAPDH was used as reference. (C) ChIP analysis of acetylated histone H4 in the human glucagon gene in BM-MSC modified by adenoviral vector (Ad) or in control non-transduced cells (NT). Position of the primer sets over the glucagon gene are indicated: GCG-207 = glucagon promoter; GCG+297 = Intron 1; GCG+3142 Exon 2. (D) Micrococcal accessibility assay of the glucagon promoter upon adenoviral transduction. DNA from BM-MSC (GFP) or adenovirus-modified BM-MSC (Pdx-1/Ngn-3/Maf-A) was extracted and digested with 1000 mU of micrococcal nuclease. Digested DNA was compared to undigested DNA by qPCR (set to 100%).</p

Efficient Generation and Amplification of High-Capacity Adeno-Associated Virus/Adenovirus Hybrid Vectors

Effective gene therapy is dependent on safe gene delivery vehicles that can achieve efficient transduction and sustained transgene expression. We are developing a hybrid viral vector system that combines in a single particle the large cloning capacity and efficient cell cycle-independent nuclear gene delivery of adenovirus (Ad) vectors with the long-term transgene expression and lack of viral genes of adeno-associated virus (AAV) vectors. The strategy being pursued relies on coupling the AAV DNA replication mechanism to the Ad encapsidation process through packaging of AAV-dependent replicative intermediates provided with Ad packaging elements into Ad capsids. The generation of these high-capacity AAV/Ad hybrid vectors takes place in Ad early region 1 (E1)-expressing cells and requires an Ad vector with E1 deleted to complement in trans both AAV helper functions and Ad structural proteins. The dependence on a replicating helper Ad vector leads to the contamination of AAV/Ad hybrid vector preparations with a large excess of helper Ad particles. This renders the further propagation and ultimate use of these gene delivery vehicles very difficult. Here, we show that Cre/loxP-mediated genetic selection against the packaging of helper Ad DNA can reduce helper Ad vector contamination by 99.98% without compromising hybrid vector rescue. This allowed amplification of high-capacity AAV/Ad hybrid vectors to high titers in a single round of propagation

Pdx-1, Ngn-3, and Maf-A expression transactivate the human insulin promoter.

<p>(A) 293T cells transduced with the LV-HIP-Luc lentivirus (left panel) and with the LV-GCG-Luc lentivirus (right panel) were infected with with LV-CMV-Pdx-1 (MOI = 1); LV-CMV-Ngn-3 (MOI = 1); LV-CMV-Maf-A (MOI = 1) or with all tree lentiviruses combined at a total MOI of 1. Luciferase activity in cell lysates was analysed 48 h post-infection. Luciferase activity of the 293T-LV-HIP-Luc and 293T_LV-GCG-Luc exposed to the lentivirus LV-CMV-GFP was set to 1. (B) Exposure of BM-MSC cells overexpressing Pdx-1, Ngn-3, and Maf-A to an adenovirus carrying an irrelevant transgene specifically induces glucagon gene expression. Human BM-MSC were transduced with LV-CMV-GFP (MOI = 2) or LV-CMV-Pdx-1 (MOI = 2); LV-CMV-Ngn-3 (MOI = 2) and LV-CMV-Maf-A (MOI = 2). Four days post- transduction, the modified cells were maintained for 3 additional days before analysis or infected with HAdV-5/fib50-EF1α-DsRed (MOI = 30). Cells were analyzed for insulin, glucagon and somatostatin gene expression by qPCR 3 days post adenovirus infection (i.e. 1 week post lentivirus infection). Data are presented as mRNA level relative to GAPDH mRNA as a reference.</p

Genes retained in E1- deleted adenoviral vector affect glucagon gene expression.

<p>(A) LV-CMV-Pdx-1 modified cells were transduced with HAdV-5/fib50-EF1α-DsRed (MOI = 30) or HD-HAdV-5/Fib50. Four days post transduction glucagon gene expression was evaluated by qPCR. GAPDH was used as reference. (B) BM-MSC(GFP) or BM-MSC (Pdx-1) were transduced with HAdV-5/fib50-EF1α-DsRed or LV-CMV-E4-ORF3 (MOI = 2). Four days post transduction glucagon gene expression was evaluated by PCR (C) or qPCR (D). GAPDH was used as reference.</p

Ectopic Pdx-1 expression in combination with HAdV-5/fib50-EF1α-DsRed infection induces glucagon gene expression in bone marrow-derived MSC.

<p>(A) BM-MSC were transduced with LV-CMV-Pdx-1 (MOI = 2), and 4 days post-transduction the modified cells were exposed to HAdV-5/fib50-EF1α-DsRed at an MOI = 30. Glucagon gene expression was evaluated by qPCR and GAPDH was used as reference. (B–C) Adenovirus transduction does not stimulate CMV-promoter driven transgene. BM-MSC were transduced with LV-CMV-GFP and increasing amounts of HAdV-5/fib50-EF1α-DsRed virus (+: MOI = 10; ++: MOI = 30). Percentage of GFP and DsRed positive cells (B) and mean fluorescent intensity (C) were evaluated by FACS (Values are indicated +/− SD). (D) Western blot analysis of LV-CMV-Pdx-1 modified cells transduced with HAdV-5/fib50-EF1α-DsRed (MOI = 30). Pdx-1 expression level was evaluated by western blot using anti-Pdx-1 antibody; anti-actin was used as loading control.</p