Molecular Therapeutic Approach Research Articles

Structural basis of pharmacological chaperoning for human β-galactosidase

GM1-gangliosidosis and Morquio B are rare lysosomal storage diseases associated with a neurodegenerative disorder or dwarfism and skeletal abnormalities, respectively. These diseases are caused by deficiencies in the lysosomal enzyme human β-Galactosidase (β-Gal), frequently related to misfolding and subsequent endoplasmic reticulum-associated degradation (ERAD) due to mutations in the β-Gal gene. Pharmacological chaperone (PC) therapy is a newly developed molecular therapeutic approach by using small molecule ligands of the mutant enzyme that are able to promote the correct folding, prevent ERAD and promote trafficking to the lysosome. Here, we present the enzymological properties of wild-type human β-Gal and two representative mutations in GM1 gangliosidosis Japanese patients (R201C and I51T). We have also evaluated the PC effect of two competitive inhibitors of β-Gal. Moreover, we determined the crystal structures of β-Gal in complex with these compouds and two structurally related analogues to elucidate the detailed atomic view of the recognition mechanism. All compounds bind to the active site of β-Gal with the sugar moiety making hydrogen bonds to active site residues. Moreover, the binding affinity, the enzyme selectivity and the PC potential are strongly affected by the mono or bicyclic structure of the core as well as the orientation, the nature and the length of the exocyclic substituent. These results provide understanding on the mechanism of action of β-Gal selective chaperoning by newly developed PC compounds.

Structural Basis of Pharmacological Chaperoning for Human β-Galactosidase

GM1 gangliosidosis and Morquio B disease are autosomal recessive diseases caused by the defect in the lysosomal β-galactosidase (β-Gal), frequently related to misfolding and subsequent endoplasmic reticulum-associated degradation. Pharmacological chaperone (PC) therapy is a newly developed molecular therapeutic approach by using small molecule ligands of the mutant enzyme that are able to promote the correct folding and prevent endoplasmic reticulum-associated degradation and promote trafficking to the lysosome. In this report, we describe the enzymological properties of purified recombinant human β-Gal(WT) and two representative mutations in GM1 gangliosidosis Japanese patients, β-Gal(R201C) and β-Gal(I51T). We have also evaluated the PC effect of two competitive inhibitors of β-Gal. Moreover, we provide a detailed atomic view of the recognition mechanism of these compounds in comparison with two structurally related analogues. All compounds bind to the active site of β-Gal with the sugar-mimicking moiety making hydrogen bonds to active site residues. Moreover, the binding affinity, the enzyme selectivity, and the PC potential are strongly affected by the mono- or bicyclic structure of the core as well as the orientation, nature, and length of the exocyclic substituent. These results provide understanding on the mechanism of action of β-Gal selective chaperoning by newly developed PC compounds.

Emerging novel concept of chaperone therapies for protein misfolding diseases.

Chaperone therapy is a newly developed molecular therapeutic approach to protein misfolding diseases. Among them we found unstable mutant enzyme proteins in a few lysosomal diseases, resulting in rapid intracellular degradation and loss of function. Active-site binding low molecular competitive inhibitors (chemical chaperones) paradoxically stabilized and enhanced the enzyme activity in somatic cells by correction of the misfolding of enzyme protein. They reached the brain through the blood-brain barrier after oral administration, and corrected pathophysiology of the disease. In addition to these inhibitory chaperones, non-competitive chaperones without inhibitory bioactivity are being developed. Furthermore molecular chaperone therapy utilizing the heat shock protein and other chaperone proteins induced by small molecules has been experimentally tried to handle abnormally accumulated proteins as a new approach particularly to neurodegenerative diseases. These three types of chaperones are promising candidates for various types of diseases, genetic or non-genetic, and neurological or non-neurological, in addition to lysosomal diseases.

Expression and mutation of the c-kit gene and correlation with prognosis of small cell lung cancer

Small cell lung cancer (SCLC) is a highly aggressive and lethal type of cancer in humans. SCLC is sensitive to chemotherapy and radiotherapy, but long-term survival is low and the majority of patients eventually develop progressive disease. With the success of imatinib mesylate in the treatment of gastrointestinal stromal tumors expressing c-kit, its use in SCLC serves as a novel molecular therapeutic approach. The activity of imatinib mesylate is correlated with the mutation of c-kit gene exons 9 and 11 in gastrointestinal stromal tumors. The incidence of epidermal growth factor receptor mutation in non-small cell lung cancer is higher in China than in the United States of America and European countries. There may be also differences in the incidence of c-kit mutation between China and European countries. At present, no study examining imatinib mesylate treatment for SCLC in China is available. To investigate the expression and mutation of c-kit and the correlation with prognosis of SCLC in China, immunohistochemistry was used to detect the expression of c-kit, and a pyrosequencing assay was used to detect mutations in c-kit exons 9 and 11 of 36 SCLC patients who received surgical treatment at the Zhejiang Cancer Hospital, Hangzhou, China, between 1998 and 2010. All 36 patients were followed up to analyze the correlation between prognosis and expression and mutation of c-kit. The incidence of c-kit-positive expression was 83.3%, including 25.0% weak staining, 22.2% moderate staining and 36.1% strong staining. The overall survival of patients with c-kit strong staining was shorter compared to patients with c-kit not strong staining. No mutation in c-kit exons 9 and 11 was detected. In conclusion, the findings showed that the expression of c-kit is high, and strong staining is a prognostic factor for worse survival.

The Potential Molecular Therapeutic Approach in Targeting Ovarian Clear Cell Carcinoma

Ovarian Clear Cell Carcinoma (OCCC) is a distinctive subtype of Epithelial Ovarian Cancer (EOC). Compared with other subtypes of EOC, CCC has relatively poor in prognosis and bad outcome in current clinical management using maximal cytoreduction and platinum plus paclitaxel–based combined chemotherapy. Therefore, the investigation of molecular therapeutic approaches targeting at signaling pathways associated with chemoresistance is needed. This review describes some recent potential signaling pathway targets and also suggests putative small molecule kinase inhibitors as well as natural anti-cancer agents in combating this disease.

HCV entry receptors as potential targets for siRNA-based inhibition of HCV

BackgroundHepatitis C virus (HCV) is a major health concern with almost 3% of the world's population (350 million individuals) and 10% of the Pakistani population chronically infected with this viral pathogen. The current therapy of interferon-α and ribavirin against HCV has limited efficiency, so alternative options are desperately needed. RNA interference (RNAi), which results in a sequence-specific degradation of HCV RNA has potential as a powerful alternative molecular therapeutic approach. Concerning viral entry, the HCV structural gene E2 is mainly involved in virus attachment to the host cell surface receptors i.e., CD81 tetraspanin, scavenger receptor class B type 1 (SR-B1), low density lipoprotein receptor (LDLR) and claudin1 (CLDN1).ResultsIn this report, we studied the relationship of the HCV receptors CD81, LDL, CLDN1 and SR-B1to HCV infection. The potential of siRNAs to inhibit HCV-3a replication in serum-infected Huh-7 cells was demonstrated by treatment with siRNAs against HCV receptors, which resulted in a significant decrease in HCV viral copy number.ConclusionsOur data clearly demonstrate that the RNAi-mediated silencing of HCV receptors is among the first of its type for the development of an effective siRNA-based therapeutic option against HCV-3a. These findings will shed further light on the possible role of receptors in inhibition of HCV-3a viral titre through siRNA mediated silencing.

Sulindac Sulfide Reverses Aberrant Self-Renewal of Progenitor Cells Induced by the AML-Associated Fusion Proteins PML/RARα and PLZF/RARα

Chromosomal translocations can lead to the formation of chimeric genes encoding fusion proteins such as PML/RARα, PLZF/RARα, and AML-1/ETO, which are able to induce and maintain acute myeloid leukemia (AML). One key mechanism in leukemogenesis is increased self renewal of leukemic stem cells via aberrant activation of the Wnt signaling pathway. Either X-RAR, PML/RARα and PLZF/RARα or AML-1/ETO activate Wnt signaling by upregulating γ-catenin and β-catenin. In a prospective study, a lower risk of leukemia was observed with aspirin use, which is consistent with numerous studies reporting an inverse association of aspirin with other cancers. Furthermore, a reduction in leukemia risk was associated with use of non-steroidal anti-inflammatory drug (NSAID), where the effects on AML risk was FAB subtype-specific. To better investigate whether NSAID treatment is effective, we used Sulindac Sulfide in X-RARα-positive progenitor cell models. Sulindac Sulfide (SSi) is a derivative of Sulindac, a NSAID known to inactivate Wnt signaling. We found that SSi downregulated both β-catenin and γ-catenin in X-RARα-expressing cells and reversed the leukemic phenotype by reducing stem cell capacity and increasing differentiation potential in X-RARα-positive HSCs. The data presented herein show that SSi inhibits the leukemic cell growth as well as hematopoietic progenitors cells (HPCs) expressing PML/RARα, and it indicates that Sulindac is a valid molecular therapeutic approach that should be further validated using in vivo leukemia models and in clinical settings.

Effect of combined siRNA of HCV E2 gene and HCV receptors against HCV

Background/AimHepatitis C virus (HCV) is a major threat as almost 3% of the world's population (350 million individual) and 10% of the Pakistani population is chronically infected with this virus. RNA interference (RNAi), a sequence-specific degradation process of RNA, has potential to be used as a powerful alternative molecular therapeutic approach in spite of the current therapy of interferon-α and ribavirin against HCV which has limited efficiency. HCV structural gene E2 is mainly involved in viral cell entry via attachment with the host cell surface receptors i.e., CD81 tetraspanin, low density lipoprotein receptor (LDLR), scavenger receptor class B type 1 (SR-B1), and Claudin1 (CLDN1). Considering the importance of HCV E2 gene and cellular receptors in virus infection and silencing effects of RNAi, the current study was designed to target the cellular and viral factors as new therapeutic options in limiting HCV infection.ResultsIn this study the potential of siRNAs to inhibit HCV-3a replication in serum-infected Huh-7 cells was investigated by combined treatment of siRNAs against the HCV E2 gene and HCV cellular receptors (CD81 and LDLR), which resulted in a significant decrease in HCV viral copy number.ConclusionFrom the current study it is concluded that the combined RNAi-mediated silencing of HCV E2 and HCV receptors is important for the development of effective siRNA-based therapeutic option against HCV-3a.

Maintenance therapy in ovarian cancer: Molecular basis and therapeutic approach

Ovarian cancer has the highest mortality rate among gynaecological tumours despite the fact that the majority of patients with advanced disease achieve complete remission after first-line surgery and chemotherapy. Unfortunately, disease recurrence occurs in the majority of patients and second-line treatments are not curative. Clearly, the persistence of dormant and drug-resistant cells after front-line treatments results in the inability to cure the disease. The identification of cancer-initiating cells or cancer stem cells as key players in the development of recurrence has opened up a novel field of research aimed at identifying additional innovative therapeutic approaches. Strategies of maintenance therapy to extend the survival of patients have been studied, but to date no overall survival benefit has been detected. Currently, numerous clinical trials have just been completed or are ongoing involving patients achieving a complete clinical response after first-line chemotherapy in order to evaluate the efficacy of different therapeutic approaches in terms of disease-free survival and overall survival. At the 2010 ASCO meeting, the first positive results of a phase III clinical trial in this setting were presented: bevacizumab (15 mg/kg i.v. every 21 days) added to first-line chemotherapy and continued for an additional 15 cycles was found to prolong progression-free survival of 3.8 months in comparison to 6 cycles of chemotherapy alone or only 6 cycles of chemotherapy plus bevacizumab. In addition, positive results were announced for a second phase III trial testing bevacizumab in the same setting, but at half dose. The final assessment of the overall clinical benefit and the approval of bevacizumab in maintenance therapy by regulatory agencies is expected to be positive, as are the final results of abagovomab phase III trial MIMOSA, another antibody-based therapy tested as a maintenance treatment for advanced ovarian cancer patients. Encouraging preliminary results confirming the safety profile and the immunogenic activity of abagovomab were presented at the last ASCO meeting. The final results are expected to be released in the first half of 2011.

Inhibition of hepatitis C virus genotype 3a by siRNAs targeting envelope genes

Hepatitis C virus (HCV) genotype 3a is considered a significant risk factor for the development of liver diseases and hepatocellular carcinoma for most of the cases in Pakistan. Because of the limited efficiency of the current therapy, RNA interference (RNAi), which results in sequence-specific degradation of HCV RNA, has potential as a powerful alternative molecular therapeutic approach. The envelope genes (E1 and E2) of HCV come in immediate contact with cells during infection and therefore might be a relevant target for new drug development. In the present study, the expression of E1 and E2 genes of HCV genotype 3a was dramatically reduced at both the mRNA and protein level using gene-specific small interfering RNAs (siRNA) when compared to mock-transfected and cells treated with control siRNAs. The potential of siRNAs to inhibit HCV-3a replication in serum-infected Huh-7 cells was also demonstrated by combined treatment of siRNAs against the E1 and E2 genes, which resulted in a significant decrease in HCV viral copy number. This clearly demonstrates that the RNAi-mediated silencing of HCV E1 and E2 is among the first of its type for the development of an effective siRNA-based therapeutic option against HCV-3a.

Oncoapoptosis: A novel molecular therapeutic for cancer treatment

Many cancer cells refractory to radiation treatment and chemotherapy proliferate due to loss of intrinsic programmed cell death (apoptosis) regulation. Consequently, the resolution of these cancers are many times outside the management capabilities of conventional therapeutics. We have developed a replication defective herpes simplex virus system which triggers apoptosis specifically in transformed human cells, termed oncoapoptosis. Susceptibility to virus induced cell death is dependent on the p53 protein status in the tumor cells, indicating specific targeting of the treatment. Primary cells which produce functional p53 are resistant to oncoapoptotic killing but not to apoptosis induced by nonviral environmental factors. Thus, induction of apoptosis by nonreplicating virus is a feasible molecular therapeutic approach for killing human cancer cells. Our findings have important implications in designing novel virus-based anticancer strategies.

Prevention of In Utero Nicotine‐Induced Bone Marrow Mesenchymal Stem Cell (BMMSC) Myofibroblast Differentation by Augmenting Lipofibroblast Phenotype

Alveolar lipofibroblasts (LIFs) are critical for lung homeostasis and injury/repair. We have shown that in utero nicotine exposure drives the differentiation of LIFs to myofibroblasts (MYFs). Whether BMMSCs can be preferentially driven to LIF phenotype is not known. We hypothesized that PPARγ agonists can prevent in utero nicotine‐induced MYF differentiation of BMMSCs and drive these cells to a LIF phenotype. Sprague Dawley dams were given 1 mg/kg nicotine from day 3 of pregnancy until term. After spontaneous delivery, pups were given either 1 or 3 mg/kg RGZ or saline (control) from postnatal day 1 to 3 weeks, when pups were sacrificed and BMMSCs were isolated and characterized for morphologic, molecular, and functional characteristics by Oil Red O staining, Western blotting, RT‐PCR, triolein uptake, and 1,2‐13 C‐glucose labeling. In utero nicotine exposure resulted in decreased Oil Red O staining, triolein uptake, oxidative/non‐oxidative ribose synthesis, PPARγ expression by BMMSCs, but markedly increased α‐SMA expression; all features, suggestive of in utero nicotine‐induced LIF‐to‐MYF transdifferentiation. Concomitant treatment with RGZ virtually blocked all of these nicotine‐induced morphologic, molecular, and functional changes, suggesting a possible targeted molecular preventive and/or therapeutic approach to prevent in utero nicotine‐induced lung injury (TRDRP 17R0170).

Molecular composition of the intercalated disc in a spontaneous canine animal model of arrhythmogenic right ventricular dysplasia/cardiomyopathy

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is characterized by ventricular arrhythmias, sudden death, and fatty or fibrofatty replacement of right ventricular myocytes. Recent studies have noted an association between human ARVD/C and molecular remodeling of intercalated disc structures. However, progress has been constrained by limitations inherent to human studies. We studied the molecular composition of the intercalated disc structure in a naturally occurring animal model of ARVD/C (Boxer dogs). We studied hearts from 12 Boxers with confirmed ARVD/C and 2 controls. Ventricular sections from 4 animals were examined by immunofluorescent microscopy. Frozen tissue samples were used for Western blot analysis. Proteins investigated were N-cadherin, plakophilin 2, desmoplakin, plakoglobin, desmin, and connexin 43 (Cx43). In control dogs, all proteins tested by immunofluorescence analysis yielded intense localized signals at sites of end-to-end cell apposition. In contrast, myocardial tissues from ARVD/C-afflicted Boxers showed preservation of N-cadherin staining but loss of detectable signal for Cx43 at the intercalated disc location. Western blots indicated that the Cx43 protein was still present in the samples. Gene sequencing analysis showed no mutations in desmoplakin, plakoglobin, Cx43, or plakophilin 2. Mutation(s) responsible for ARVD/C in Boxers lead, directly or indirectly, to severe modifications of mechanical and electrical cell-cell interactions. Furthermore, significant reduction in gap junction formation may promote a substrate for malignant ventricular arrhythmias. This model may help to advance our understanding of the molecular basis, pathophysiology, and potential therapeutic approach to patients with ARVD/C.

Uncommon tumors and exceptional therapies: paradox or paradigm?

Why does it seem that, repeatedly, when a new treatment with a striking effect is discovered in the cancer field, it is effective for a very rare cancer type? For example, groundbreaking therapeutic discoveries have been made for extremely uncommon malignancies such as hairy cell leukemia, chronic myelogenous leukemia, seminoma, gastrointestinal stromal tumor, (del)5q myelodysplastic syndrome, and acute promyelocytic leukemia. In contrast, progress in the most common and most intensively studied tumors - lung, breast, prostate, and colon cancer - has been slow and incremental. We hypothesize that the reason for this phenomenon is that the pathophysiologic basis for a tumor being rare is one and the same as the reason that it may ultimately be so treatable. That is, if a cancer can be derived only via a single aberrant molecular genetic aberration, then it should be both rare and easily targeted by a molecular cancer therapeutic approach. If, on the other hand, many distinct pathways can lead to the development of a specific tumor type, it should occur much more commonly and be significantly more difficult to treat. The corollary to our hypothesis is the prediction that new therapies will continue to show their most salutary effects in rare cancers. Furthermore, only by stratifying the common tumors, especially when using targeted agents, into the molecular subsets of diseases that compose them are we likely to achieve a substantial effect in these disorders.

β‐Galactosidase deficiency: An approach to chaperone therapy

We propose a new molecular therapeutic approach to lysosomal diseases with severe neurological manifestations. Some low-molecular-weight compounds, acting as competitive inhibitors of a lysosomal enzyme in vitro, were found to stabilize and restore catalytic activities of the enzyme molecule as a molecular chaperone. We started this trial first in Fabry disease (generalized vasculopathy) using galactose and 1-deoxygalactonojirimycin, and then in beta-galactosidase deficiency disorders (beta-galactosidosis) with generalized neurosomatic and/or systemic skeletal manifestations (GM(1)-gangliosidosis and Morquio B disease), using a newly developed chemical compound N-octyl-4-epi-beta-valienamine (NOEV). Administration of this chaperone compound resulted in elevation of intracellular enzyme activity in cultured fibroblasts from patients and genetically engineered model mice. In addition, substrate storage was improved after NOEV had been transported into the brain tissue via the blood-brain barrier. We hope this new approach (chemical chaperone therapy) will be useful for certain patients with beta-galactosidosis and potentially other lysosomal storage diseases with central nervous system involvement.

Role of Extracellular Signal-Regulated Kinase-1/2 (ERK) on Complete Remission Achievement of Primary Adult Acute Lymphoblastic Leukemia Patients Enrolled in the GIMEMA Protocol LAL 2000: In Vitro Activity of the MEK Inhibithor PD98059.

The mitogen-activated protein kinase (MAPK) pathway contributes to the proliferative and differentiative control of hematopoietic cells. Extracellular signal-regulated kinase-1/2 (ERK) is one of the members of the MAPK pathway; its constitutive activation (p-ERK) has been found in a broad spectrum of hematological malignancies, particularly in acute myeloid leukemia (AML). A molecular therapeutic approach based on MAPK inhibitors has been therefore attempted. The aim of this study was to assess the expression and the clinical role of p-ERK in adult acute lymphoblastic leukemia (ALL) evaluating, in addition, the in vitro effects of the MEK inhibitor PD98059, used alone or in combination with chemotherapy, on the proliferation and apoptosis of ALL blast cells. Primary samples from 131 uniformly treated de novo patients enrolled in the GIMEMA LAL 2000 protocol were evaluated, using a p-ERK1/2-specific MoAb (clone E10), by Western blot analysis and flow cytometric assay which allowed single cell phospho-protein determination by the Kolmogorov-Smirnov statistic test (D-value). Lymphoid cell lines (Jurkat, CEM, RAJI and RPMI8866), as well as normal PMA-activated PBL expressed p-ERK, whereas resting PBL exhibited minimal levels (D-value <0.10). In clinical ALL samples p-ERK ranged between 0 and 0.83, constitutive activation (D-value ≥ 0.10) was found in 45/131 samples (34.5%) and resulted significantly associated with higher WBC values (>50x109/L; P=0.0128) and lower RNA-index of G1 phase (P=0.0296). Age, leukemia phenotype and molecular characteristics did not associate with p-ERK expression, while phospho-protein levels, measured as a continuous variable, correlated with failure to achieve CR (P=0.069). According to cell availability, we evaluated the effects of the MEK inhibitor PD98059 at 25μM on 30 fresh ALL samples, cultured in vitro for 24 hours. Eight of them were characterized by constitutive ERK activation, which was efficiently abrogated in 6; however, this effect did not induce cell cycle changes or apoptosis either alone or in combination with chemotherapeutic agents (Ara-C and/or Dexametasone). In summary, our study demonstrated that p-ERK is expressed in one third of adult ALL samples at presentation. Patients with p-ERK expression are characterized by higher leukemic mass, with a trend to induction treatment failure. MEK inhibition by PD98059 is capable of modulating p-ERK in primary ALL cells, though it does not result in cell cycle/apoptotic changes. In conclusion, ERK signaling appears only partially involved in the control of proliferative signals in adult ALL and modulation of additional pathways may result in more effective therapeutic strategies.

Silencing pathways that underlie platinum resistance in ovarian cancer cells using small interfering RNA

5117 Background: Primary therapy for women with advanced stage epithelial ovarian cancer includes primary surgical cytoreduction followed by platinum-based chemotherapy. Although nearly 70% of patients demonstrate a complete clinical response to primary therapy, the majority of patients will develop platinum-resistant recurrent disease. Our understanding of this chemo-resistance remains incomplete. The expression of genes such as c-myc have been shown to be associated with chemoresistance in several human cancers. Recently, using microarray expression analysis of 55 primary epithelial ovarian cancers, we demonstrated that members of the c-myc pathway are predictive of response to primary platinum therapy. We hypothesized that increased c-myc gene expression also underlies platinum resistance in ovarian cancer cell lines, and that it can be reduced by use of a c-myc-targeted small interfering RNA (si-cmyc) approach, thus reversing platinum resistance. Methods: Expression of the c-myc gene was measured in platinum-sensitive (OV2008) mother and platinum-resistant (C13) daughter ovarian cancer cell lines using quantitative real time polymerase chain reaction (QRTPCR). Platinum-resistant cells were then subject to c-myc-targeted small interfering RNA (50nM, 100nM and 150nM), and c-myc gene expression was evaluated using Western blot analysis. Platinum-resistant ovarian cancer cells, subject to successful c-myc expression silencing by siRNA, were then evaluated for platinum-response using the MMT assay. Results: Expression of the c-myc gene was 1.78-fold higher in platinum-resistant versus platinum-sensitive ovarian cancer cells. Western blot analysis demonstrated a reduction of c-myc gene expression by 95% in platinum-resistant ovarian cancer cells subject to c-myc-targeted siRNA at 150nM. Conclusions: These findings support the hypothesis that the c-myc gene pathway is an important determinant of platinum-response in ovarian cancer cells. Further, we have demonstrated that siRNA approaches can selectively silence genes that underlie platinum-response and may thus represent a novel targeted molecular therapeutic approach to patients with platinum-resistant ovarian cancer. No significant financial relationships to disclose.

Inhibition of HIV-1 fusion with small interfering RNAs targeting the chemokine coreceptor CXCR4.

RNA interference (RNAi) is an evolutionarily conserved process by which plants and animals protect their genomes utilizing small, double-stranded RNAs to degrade target RNAs in a sequence-specific manner. Post-transcriptional gene silencing by these moieties can lead to degradation of both cellular and viral RNAs. It has recently been shown that double-stranded, small interfering RNAs (siRNAs) of 21-25 nucleotides can be transfected into relevant cells to target specific RNAs. This approach was utilized to inhibit human immunodeficiency virus type I (HIV-1) infection in human cells. siRNAs with homology to a motif in the mRNA that encodes for the HIV-1 chemokine coreceptor CXCR4 was utilized. Complementary studies via immunofluorescence microscopy and fluorescence-activated cell sorting demonstrated downregulation of CXCR4 from the surface of cells transfected with the specific siRNAs. As well, siRNAs without sequence homology to CXCR4 were used as controls and demonstrated no downregulation of CXCR4. siRNAs targeted to another chemokine coreceptor, APJ, showed specificity for downregulation of APJ but had no effects on CXCR4. Transfections with siRNAs targeting CXCR4 mRNA were shown to inhibit HIV-1 envelope fusion, which is relatively resistant to most viral inhibitors targeting chemokine coreceptors. The specificity of this effect was demonstrated by the inhibition of fusion by CXCR4-tropic and dual-tropic (CXCR4 and CCR5) envelope glycoproteins from HIV-1 on CXCR4+ indicator cells, but the lack of effects by siRNAs targeting CXCR4 mRNA on dual-tropic HIV-1 envelopes in CCR5+ indicator cells utilizing these fusion assays. Interestingly, siRNAs targeting CXCR4 selectively inhibited CXCR4-tropic cell-free virus infection of human cells but at only modest levels as compared to cell:cell fusion. siRNA may be a potential molecular therapeutic approach to alter a cellular cofactor critical for infection of human cells by relevant strains of HIV-1. The targeting of a cellular cofactor, rather than the HIV-1-specific mRNAs or genomic RNA, holds promise as the rapid mutational ability of the HIV-1 genome may obviate the potential clinical use of RNAi directly against this virus.

Developing aptamers into therapeutics.

The idea of using nucleic acid molecules as therapeutic agents was conceived in the 1970s with the development of antisense strategies. Antisense compounds are single-stranded nucleic acids that, in principle, disrupt the synthesis of a targeted protein by hybridizing in a sequence-dependent manner to the mRNAs that encode it. The mechanism of inhibition by nucleic acid aptamers is fundamentally different. Aptamers are single-stranded nucleic acids that directly inhibit a protein’s function by folding into a specific three-dimensional structure that dictates high-affinity binding to the targeted protein. Through iterative in vitro selection techniques, aptamers can be generated that bind essentially any protein (or small molecule) target. A high-affinity, specific inhibitor can theoretically be made to order, provided that a small quantity of pure target is available. Because they inhibit the activity of existing proteins directly, aptamers are more similar to monoclonal antibody or small molecule drugs than to antisense compounds, and this property greatly increases the number of clinical indications that are potentially treatable by nucleic acid–based compounds. Aptamers have been generated against a wide variety of targets, a complete discussion of which is beyond the scope of this article (for review, see ref. 1). As with any molecular therapeutic approach, the inhibitor is only as effective as the target is important. Through their ability to specifically inhibit a molecule of interest, aptamers have already proven useful as reagents for target validation in a variety of disease models. The next step, therapeutic utility, will depend on the efficacy of these novel compounds in humans. In this Perspective, we will focus on the development and status of aptamers as therapeutic molecules.

Novel molecular therapeutic approach to cardiovascular disease based on hepatocyte growth factor.

Gene therapy techniques are being developed as potential treatments for cardiovascular diseases. During the past decade, many gene transfer methods including viral transfer techniques have been developed, and some are being applied clinically in human gene therapy studies. Recently, we have developed a novel gene transfer method mediated by Hemagglutinating Virus of Japan (HVJ) liposome, with which we have already reported several cases of successful gene transfer in vivo. Since the virus is inactivated by ultraviolet light, there is little potential for biological hazard with this method as compared to other viral gene transfer approaches. We also developed a novel strategy of gene therapy for cardiovascular diseases utilizing hepatocyte growth factor (HGF) which is an endothelial cell specific growth factor and an angiogenic growth factor. Based on these facts, we hypothesized that HGF may prevent restenosis after angioplasty through re endothelialization and myocardial infarction through induction of angiogenesis. The present results provide evidence of the efficacy of supplemental therapy with HGF by gene transfer in cardiovascular diseases. These data suggest the efficacy of novel molecular therapeutic approaches as gene therapy for cardiovascular diseases such as restenosis and myocardial infarction.