Assessment of the Oral Delivery of a Myelin Basic Protein Gene Promoter with Antiapoptotic bcl-xL (pMBP-bcl-xL) DNA by Cyclic Peptide Nanotubes with Two Aspect Ratios and Its Biodistribution in the Brain and Spinal Cord.

Cyclo-(D-Trp-Tyr) peptide nanotubes (PNTs) were reported to be potential carriers for oral gene delivery in our previous study; however, the effect of the aspect ratio (AR) of these PNTs on gene delivery in vivo could affect penetration or interception in biological environments. The aim of this study was to assess the feasibility of cyclo-(D-Trp-Tyr) PNTs with two ARs as carriers for oral pMBP-bcl-xL-hRluc delivery to the spinal cord to treat spinal cord injury (SCI). We evaluated the biodistribution of oligodendrocyte (OLG)-specific myelin basic protein gene promoter-driven antiapoptotic DNA (pMBP-bcl-xL) to the brain and spinal cord delivered with cyclo-(D-Trp-Tyr) PNTs with large (L) and small (S) PNTs with two ARs. After complex formation, the length, width, and AR of the L-PNTs/DNA were 77.86 ± 3.30, 6.51 ± 0.28, and 13.75 ± 7.29 µm, respectively, and the length and width of the S-PNTs/DNA were 1.17 ± 0.52 and 0.17 ± 0.05 µm, respectively, giving an AR of 7.12 ± 3.17 as detected by scanning electron microscopy. Each of these three parameters exhibited significant differences (p < 0.05) between L-PNTs/DNA and S-PNTs/DNA. However, there were no significant differences (p > 0.05) between the L-PNTs and S-PNTs for either their DNA encapsulation efficiency (29.72 ± 14.19 and 34.31 ± 16.78%, respectively) or loading efficiency (5.15 ± 2.58 and 5.95 ± 2.91%). The results of the in vitro analysis showed that the S-PNT/DNA complexes had a significantly higher DNA release rate and DNA permeation in the duodenum than the L-PNT/DNA complexes. Using Cy5 and TM-rhodamine to individually and chemically conjugate the PNTs with plasmid DNA, we observed, using laser confocal microscopy, that the PNTs and DNA colocalized in complexes. We further confirmed the complexation between DNA and the PNTs using fluorescence resonance energy transfer (FRET). Data from an in vivo imaging system (IVIS) showed that there was no significant difference (p > 0.05) in PNT distribution between L-PNTs/DNA and S-PNTs/DNA within 4 h. However, the S-PNT/DNA group had a significantly higher DNA distribution (p < 0.05) in several organs, including the ilium, heart, lungs, spleen, kidneys, testes, brain, and spinal cord. Finally, we determined the bcl-xL protein expression levels in the brain and spinal cord regions for the L-PNT/DNA and S-PNT/DNA complex formulations. These results suggested that either L-PNTs or S-PNTs may be used as potential carriers for oral gene delivery to treat SCI.

Effects of long-term hypoxia and pro-survival cocktail in bone marrow-derived stromal cell survival.

The goal of this study was to determine whether a pro-survival cocktail (PSC, consisting of IGF-1, Bcl-XL, and Caspase-I Inhibitor) and long-term hypoxia (LTH) enhance survival and functional properties of bone marrow-derived stromal stem cells (BMSCs), in response to stress conditions. PSC-treated cells retained BMSC surface markers and protected cells from apoptosis under serum starvation and ischemic (1% O2 and 100 µM H2O2) conditions. LTH promoted osteogenesis, while suppressing adipogenesis. LTH alone did not result in an improvement in the apoptosis rate; however, PSC conferred significant protection regardless of the oxygenation status. One of the possible mechanisms of PSC protection was due to the elevated phospho-AKT in treated groups. PSC treatment or LTH did not alter migration toward stem cell-derived factor-1 alpha (SDF-1α) or fetal bovine serum, nor did they enhance cell motility during wound healing. There was no difference in the secreted cytokine profiles of BMSCs treated with PSC after stress when grown in normoxic or LTH. However, LTH did upregulate the vascular endothelial growth factor, hepatocyte growth factor, and SDF-1α, while it downregulated other anti- and proinflammatory cytokines and chemokines. We also observed a high degree of interdonor BMSC variability in response to pretreatment with PSC and LTH, confounding the functional results, underscoring the observation that not all donor-derived BMSCs will respond similarly.

Automated microinjection of recombinant BCL-X into mouse zygotes enhances embryo development.

Progression of fertilized mammalian oocytes through cleavage, blastocyst formation and implantation depends on successful implementation of the developmental program, which becomes established during oogenesis. The identification of ooplasmic factors, which are responsible for successful embryo development, is thus crucial in designing possible molecular therapies for infertility intervention. However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos. We have developed an automated robotic microinjection system for delivering cell impermeable compounds into preimplantation embryos with a high post-injection survival rate. In this paper, we report the performance of the system on microinjection of mouse embryos. Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL) protein is effective in preventing early embryo arrest imposed by suboptimal culture environment. We demonstrate that microinjection of recBCL-XL protein into early-stage embryos repairs mitochondrial bioenergetics, prevents reactive oxygen species (ROS) accumulation, and enhances preimplantation embryo development. This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF) failure due to poor embryo quality.

Evaluation of apoptotic and anti-apoptotic genes on efficacy of DNA vaccine encoding glycoprotein B of Herpes Simplex Virus type 1.

Many approaches have so far been tried to enhance the immunogenicity of DNA vaccine. These include the use of various factors that induce apoptosis or anti-apoptosis effects when co-delivered with DNA vaccine. In the present study, the effects of pro-apoptotic Bax encoding plasmid (pBax) and anti-apoptotic Bcl-X(L) encoding plasmid (pBcl-xl), intradermally co-injected with glycoprotein B (gB) of Herpes Simplex Virus (HSV)-1 encoding plasmid (pgB) into the C57BL/6 mice were evaluated. Immune responses of the mice to the antigen were assessed by antibody assay, lymphoproliferative responses as well as cytokine and cytotoxic T-lymphocyte (CTL) assay. Analysis of the humoral and cellular responses showed that the mice immunized with pBax and pgB induced higher levels of antibody and Interleukin-4 as well as stronger lymphocyte proliferative responses and cytotoxic activity compared to those mice received pgB alone. pBcl-xl when intradermally co-injected with pgB showed no significant enhancement in immune responses comparing to pgB.

Gene transfection to retinal ganglion cells mediated by ultrasound microbubbles in vitro.

RATIONALE AND OBJECTIVES: To investigate the expression levels of green fluorescence protein (GFP) into retinal ganglion cells (RGCs) in vitro by ultrasound-mediated microbubble destruction (UMMD) and assess the effect of bcl-xl gene on N-methyl-D-aspartate (NMDA)-induced apoptosis in the cultured RGCs by UMMD. MATERIALS AND METHODS: pEGFP-N1 was transfected to RGCs in vitro by UMMD and liposome was used as the control. The transfection effect was detected using microscope and flow cytometry qualitatively and quantitatively. Monotetrazolium was adopted to measure the cell vitality. NMDA was used to induce apoptosis in the cultured RGCs, and the bcl-xl gene was transfected into RGCs by UMMD before NMDA-induced apoptosis. The expression of bcl-xl protein in RGCs was assessed by immunohistochemistry assay. The amorphous character of RGCs was revealed by acridine orange and ethidium bromide staining. DNA fragment was detected by agarose gel electrophoresis. RESULTS: Ultrasound combined with microbubbles enhanced gene transfection to the cultured cells in some condition. The average transfection rate of pEGFP-N1 with UMMD was 25%. Both ultrasound and microbubble had no effect on cell viability. The expression of bcl-xl protein in transfected and non-transfected RGCs was significantly different. Less apoptotic bodies and no representative DNA fragment were detected in the treatment group. CONCLUSIONS: Microbubble destruction can enhance the reporter gene transfection and expression and have a good target. Transfection of bcl-xl gene has an anti-apoptosis effect on the cultured RGCs induced by NMDA with UMMD.

Fusion of the tetanus toxin C fragment binding domain and Bcl-xL for protection of peripheral nerve neurons.

OBJECTIVE: Apoptosis has been shown to play an important role in motor neuron (MN) degeneration in both neurodegenerative disease and peripheral neuropathy. Bcl-xL, an antiapoptotic protein, is down-regulated in these etiologies [corrected] The carboxyl-terminal domain of the tetanus toxin heavy chain (Hc) has high affinity for axon terminal binding and uptake into motor and dorsal root ganglion (DRG) neurons. We report the development of a fusion protein between Hc and Bcl-xL to enhance uptake of Bcl-xL by MNs as a strategy for inhibiting peripheral neuronal apoptosis. METHODS: The genes for Hc, Bcl-xL, and green fluorescent protein were cloned into an Escherichia coli expression system in 2 different arrangements. Fusion proteins were purified through chromatography. Cultured E15 rat spinal cord MNs and DRG cells were used to demonstrate neuron-specific uptake and retrograde transport of the fusion proteins mediated by Hc. Finally, glutamate-induced apoptosis was used as an in vitro model to measure the antiapoptotic effects of the fusion proteins. RESULTS: Bcl-xL fusion proteins were found to bind specifically and undergo uptake into cultured rat spinal MNs. The fusion proteins were also taken up by DRG axonal terminals and transported back to the cell bodies in Campenot compartmentalized chambers (Tyler Research Corp., Edmonton, Canada). Finally, fusion protein application improved cell survival and decreased apoptosis in glutamate-mediated excitotoxicity of the SH-SY5Y neuronal cells. CONCLUSION: Hc can be applied as a universal carrier for therapeutic cargo delivery specifically to MNs or DRGs. The fusion proteins between Bcl-xL and Hc constructed in this study might bear applications to the treatment of MN disease, neuropathy, or nerve injury through nerve or intramuscular injection.

Therapeutic benefits of intrathecal protein therapy in a mouse model of amyotrophic lateral sclerosis.

When fused with the protein transduction domain (PTD) derived from the human immunodeficiency virus TAT protein, proteins can cross the blood-brain barrier and cell membrane and transfer into several tissues, including the brain, making protein therapy feasible for various neurological disorders. We have constructed a powerful antiapoptotic modified Bcl-X(L) protein (originally constructed from Bcl-X(L)) fused with PTD derived from TAT (TAT-modified Bcl-X(L)), and, to examine its clinical effectiveness in a mouse model of familial amyotrophic lateral sclerosis (ALS), transgenic mice expressing human Cu/Zn superoxide dismutase (SOD1) bearing a G93A mutation were treated by intrathecal infusion of TAT-modified Bcl-X(L). We demonstrate that intrathecally infused TAT-fused protein was effectively transferred into spinal cord neurons, including motor neurons, and that intrathecal infusion of TAT-modified Bcl-X(L) delayed disease onset, prolonged survival, and improved motor performance. Histological studies show an attenuation of motor neuron loss and a decrease in the number of cleaved caspase 9-, cleaved caspase 3-, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in the lumbar cords of TAT-modified Bcl-X(L)-treated G93A mice. Our results indicate that intrathecal protein therapy using a TAT-fused protein is an effective clinical tool for the treatment of ALS.

Bcl-xL antisense oligonucleotide and cisplatin combination therapy extends survival in SCID mice with established mesothelioma xenografts.

Bcl-xL functions as a dominant regulator of apoptotic cell death and is implicated in chemotherapeutic resistance of malignant pleural mesothelioma (MPM). Mesothelioma cell lines demonstrate increasing levels of Bcl-xL as resistant clones are selected in vitro. Moreover, upon introduction of antisense oligonucleotides specific to Bcl-xL mRNA, MPM cells are sensitized to chemotherapeutic agents. Here we describe the therapeutic effects of a novel combination therapy, Bcl-xL antisense oligonucleotide (ASO 15999) and cisplatin, on mesothelioma cell lines in vitro and in vivo; in addition, efficacy of ASO 15999 in decreasing tumor load as well as its effect on survival in an animal model. Finally, we initiated preliminary toxicity studies involved with intraperitoneal (IP) injections of ASO 15999 into mice. This novel combination, with doses of cisplatin four times below established IC(50) levels, significantly decreased viability of MPM cell lines after 48 hr. The growth of established mouse flank human tumor xenografts was reduced with intra-tumor administration of ASO 15999. Local spread and development of IP xenografts was reduced with treatments of ASO alone, and survival of mice afflicted with these xenografts was prolonged after administration of ASO alone and ASO 15999 + cisplatin combination therapy. These findings suggest that ASO 15999 sensitizes MPM cell lines to the toxic effects of cisplatin. ASO 15999 induced reduction of Bcl-xL is effective in slowing the progression of human mesothelioma cell lines both in vitro and in vivo. More notably, the combination of Bcl-xL ASO and cisplatin extends survival in an orthotopic tumor xenograft model.

Detrimental effects of antiapoptotic treatments in spinal cord injury.

Long-term functional impairments due to spinal cord injury (SCI) in the rat result from secondary apoptotic death regulated, in part, by SCI-induced decreases in protein levels of the antiapoptotic protein Bcl-xL. We have shown that exogenous administration of Bcl-xL spares neurons 24 h after SCI. However, long-term effects of chronic application of Bcl-xL have not been characterized. To counteract SCI-induced decreases in Bcl-xL and resulting apoptosis, we used the TAT protein transduction domain fused to the Bcl-xL protein (Tat-Bcl-xL), or its antiapoptotic domain BH4 (Tat-BH4). We used intrathecal delivery of Tat-Bcl-xL, or Tat-BH4, into injured spinal cords for 24 h or 7 days, and apoptosis, neuronal death and locomotor recovery were assessed up to 2 months after injury. Both, Tat-Bcl-xL and Tat-BH4, significantly decreased SCI-induced apoptosis in thoracic segments containing the site of injury (T10) at 24 h or 7 days after SCI. However, the 7-day delivery of Tat-Bcl-xL, or Tat-BH4, also induced a significant impairment of locomotor recovery that lasted beyond the drug delivery time. We found that the 7-day administration of Tat-Bcl-xL, or Tat-BH4, significantly increased non-apoptotic neuronal loss and robustly augmented microglia/macrophage activation. These results indicate that the antiapoptotic treatment targeting Bcl-xL shifts neuronal apoptosis to necrosis, increases the inflammatory response and impairs locomotor recovery. Our results suggest that a combinatorial treatment consisting of antiapoptotic and anti-inflammatory agents may be necessary to achieve tissue preservation and significant improvement in functional recovery after SCI.

Microdevice-based delivery of gene products using sonoporation.

This paper presents a proof-of-concept miniature device for delivery of antisense oligonucleotides (ASO). A piezoelectric, lead zirconate titanate (PZT) plate (0.5 cm(2) x 0.75 mm) is used to transfect cells using cavitation-induced sonoporation. Both human umbilical vein endothelial cells (HUVEC) and human prostate cancer cells (PC3) are investigated in vitro. Preliminary results show that after sonication, the transfection rate for HUVEC increases by 96% compared to controls (p < 0.01). For PC3, the transfection rate increases by 31% compared to controls (p < 0.02). This research can potentially be applied in realizing a microelectromechanical system (MEMS)-based device for gene therapy in cancer treatment.

TAT-mediated delivery of Bcl-xL protein is neuroprotective against neonatal hypoxic-ischemic brain injury via inhibition of caspases and AIF.

Systemic delivery of recombinant Bcl-xL fusion protein containing the TAT protein transduction domain attenuated neonatal brain damage following hypoxic ischemia (H-I). Within 30 min after intraperitoneal injection of TAT-Bcl-xL protein into 7-day-old rats, substantially enhanced levels of Bcl-xL were found in several brain regions. Administration of TAT-Bcl-xL at the conclusion of the H-I insult decreased cerebral tissue loss in a dose-dependent manner measured 1 and 8 weeks later. Neuroprotection provided by TAT-Bcl-xL was significantly greater than that of the pan-caspase inhibitor BAF, suggesting that protection is only partially attributable to caspase inhibition by TAT-Bcl-xL. TAT-Bcl-xL not only inhibited caspases-3 and -9 activities after H-I but also prevented nuclear translocation of AIF. Taken together, these results substantiate the feasibility of peripheral delivery of an anti-apoptotic factor into the brain of neonatal animals to reduce H-I-induced brain injury.

[A study on the effects of rAd-gfp-bcl-X(L) on retinal degeneration in mice].

OBJECTIVE: To observe the therapeutic effect of rAd-gfp-bcl-X(L) on retinal degeneration (RD) in mice. METHODS: rAd-gfp-bcl-X(L) granules were injected into the subretinal space of right eyes of RD mice in Group A (postnatal days 10, p10) and Group B (postnatal days 22, p22) (n = 20). The morphological changes were observed by light microscopy and transmission electron microscopy at 15 - 30 days post-injection. Frozen section was used to observe the expression of GFP. The level of bcl-X(L) mRNA and bcl-X(L) protein were assayed by RT-PCR and immunohistochemistry assay. RESULTS: In frozen section, GFP was observed in the treated eyes under fluorescence microscopy, indicating that rAd-gfp-bcl-X(L) had transfected into the retinal cells successfully and that bcl-X(L) was expressed. In Group A, an increased expression of bcl-X(L) mRNA and Bcl-X(L) protein were observed in the treated eyes. The outer nuclear layer of the treated eyes in Group A was thicker than that of the control eyes at 30 days post-injection. The inner segment of treated eyes in Group A was healthier than that of control eyes, as indicated by transmission electron microscopy. No difference was found in the thickness of retina between treated eyes and control eyes in Group B. CONCLUSION: The bcl-X(L) can be expressed and have an anti-apoptosis effect when the rAd-gfp-bcl-X(L) particles are injected into the subretinal space of RD mice's eyes at an early stage. But the expression and protective effects were not observed in RD mice at a later stage. The therapy of rAd-gfp-bcl-X(L) for retinal degenerative diseases still needs further study.