After two decades of ups and downs, gene therapy has recently achieved a milestone in treating patients with Leber's congenital amaurosis (LCA). LCA is a group of inherited blinding diseases with retinal degeneration and severe vision loss in early infancy. Mutations in several genes, including RPE65, cause the disease. Using adeno-associated virus as a vector, three independent teams of investigators have recently shown that RPE65 can be delivered to retinal pigment epithelial cells of LCA patients by subretinal injections resulting in clinical benefits without side effects. However, considering the whole field of gene therapy, there are still major obstacles to clinical applications for other diseases. These obstacles include innate and immune barriers to vector delivery, toxicity of vectors and the lack of sustained therapeutic gene expression. Therefore, new strategies are needed to overcome these hurdles for achieving safe and effective gene therapy. In this article, we shall review the major advancements over the past two decades and, using lung gene therapy as an example, discuss the current obstacles and possible solutions to provide a roadmap for future gene therapy research.
Adaptive Immunity ; Carrier Proteins ; genetics ; Cystic Fibrosis ; genetics ; therapy ; Cystic Fibrosis Transmembrane Conductance Regulator ; genetics ; Dependovirus ; genetics ; Eye Proteins ; genetics ; Gene Targeting ; Genetic Therapy ; methods ; Genetic Vectors ; Humans ; Immunity, Innate ; Leber Congenital Amaurosis ; genetics ; therapy ; Liposomes ; Lung ; drug effects ; metabolism ; pathology ; Mutation ; Retina ; drug effects ; metabolism ; pathology ; Retroviridae ; genetics ; cis-trans-Isomerases

Animals ; Brain ; metabolism ; Humans ; Nerve Regeneration ; drug effects ; Neurodegenerative Diseases ; metabolism ; prevention & control ; Neuroprotective Agents ; metabolism ; therapeutic use ; Tacrolimus Binding Protein 1A ; metabolism ; Tacrolimus Binding Proteins ; metabolism ; therapeutic use

AIMTo identify recombinant protein rhFKBP12 by new technique ESI-quadrupole-oa-TOF tandem mass spectrometry.

METHODSThe molecular weight of rhFKBP12 was measured by ESI-MS. Digest rhFKBP12 by trypsin at 37 degrees C over night. The tryptic digested peptides were measured and then two doublely charged peptides were selected to measure their amino acid sequence by ESI-MS/MS. Search database with the measured amino acid sequence to identify rhFKBP12.

RESULTSThe calculated molecular weight of rhFKBP12 was 11,819.54 and the measured value was 11,820.38. The measurement percent error was only 0.007%. The sequence measured by ESI-MS/MS was QVETMS and EEGVAQMSV and then the database search results with them were both hFKBP12.

CONCLUSIONThe study proves that the primary structure of rhFKBP12 is correct and there is no amino acid deletion, mutation and modification in its expression, refolding and purification. It also shows that ESI-MS/MS is a good method to identify protein with advantage of sensitivity, high speed and accuracy.

Amino Acid Sequence ; Molecular Weight ; Peptide Mapping ; Recombinant Proteins ; chemistry ; isolation & purification ; Spectrometry, Mass, Electrospray Ionization ; methods ; Tacrolimus Binding Protein 1A ; chemistry ; isolation & purification

BACKGROUND AND OBJECTIVES: The FK-506 binding protein 12 (FKBP12) regulates intracellular Ca2+ release by stabilizing the Ca2+-induced Ca2+-release channel (ryanodine receptor) in skeletal muscle. It has been recently shown that a different FKBP, FKBP12.6, is specifically associated with cardiac ryanodine receptor. Since the role of FKBP12.6 in excitation-contraction coupling in the cardiac muscle has not been precisely determined, its biological function was assessed and expression patterns of FKBP12.6 were evaluated in the various models of heart disease. MATERIAL AND METHODS: The mouse (m) FKBP12.6 gene was cloned and characterized after screening a mouse genomic DNA library using a mFKBP12.6 cDNA obtained through reverse transcriptase-polymerase chain reaction. Expression levels of mFKBP12.6 was evaluated during cardiac development and in the models of cardiac hypertrophy and failure. RESULTS: Both mFKBP12.6 and mFKBP12 contain an open reading frame of 327 nucleotides encoding 108 amino acids. Comparison of mFKBP12.6 cDNA to rat FKBP12.6, human FKBP12.6 and mFKBP12 cDNA revealed 95%, 94% and 74% identity in nucleotide sequence and 98%, 97% and 80% identity in amino acid sequence, respectively. Purified recombinant mFKBP12.6 migrated slower than either mFKBP12 or human FKBP12 on an SDS-polyacrylamide gel, despite having the same number of amino acids and a slightly lower calculated molecular mass. Northern blot analysis showed that the expression of FKBP12 and FKBP12.6 to be highest in brain. While the expression of FKBP12 was much stronger in adult than in embryonic hearts, it was further increased following pressure overload hypertrophy. FKBP12.6 mRNA expression analyzed by RNase protection assay was upregulated after induction of cardiac hypertrophy like FKBP12, whereas it was decreased in the failing heart. The mFKBP12.6 gene contains 5 exons and the proteincoding region of the gene was divided into 4 exon modules. CONCLUSION: We report the molecular cloning and characterization of the mouse FKBP12.6 gene. According to these results, FKBP12 and FKBP12.6 may play a role in the development of cardiac hypertrophy and transition to heart failure. To precisely determine the role of FKBP12 and FKBP12.6 in the heart, a strategy using homologous recombination in embryonic stem cells to conditionally ablate exon 2 of mFKBP12.6 gene has been developed and initial characterization is now underway.
Adult ; Amino Acid Sequence ; Amino Acids ; Animals ; Base Sequence ; Blotting, Northern ; Brain ; Cardiomegaly ; Carrier Proteins* ; Clone Cells ; Cloning, Molecular* ; Cloning, Organism ; DNA, Complementary ; Embryonic Stem Cells ; Exons ; Gene Library ; Heart Diseases* ; Heart Failure ; Heart* ; Homologous Recombination ; Humans ; Hypertrophy ; Mass Screening ; Mice* ; Muscle, Skeletal ; Myocardium ; Nucleotides ; Open Reading Frames ; Rats ; Ribonucleases ; RNA, Messenger ; Ryanodine Receptor Calcium Release Channel ; Tacrolimus Binding Protein 1A ; Tacrolimus Binding Proteins ; Tacrolimus*

Cyclophilin A (CyPA) is a 17 kDa, ubiquitously expressed multifunctional protein that possesses peptidylprolyl cis-trans isomerase activity and scaffold function. Its expression is increased in inflammatory conditions including rheumatoid arthritis, autoimmune disease and cancer. Intracellular CyPA regulates protein trafficking, signal transduction, transcription regulation and the activity of certain other proteins. Secreted CyPA activates cardiovascular cells resulting in a variety of cardiovascular diseases; including vascular remodeling, abdominal aortic aneurysms formation, atherosclerosis, cardiac hypertrophy and myocardial ischemic reperfusion injury.
Aortic Aneurysm, Abdominal ; Arthritis, Rheumatoid ; Atherosclerosis ; Autoimmune Diseases ; Cardiomegaly ; Cardiovascular Diseases ; Cyclophilin A ; Cyclophilins ; Myocardial Reperfusion Injury ; Oxidative Stress ; Protein Transport ; Proteins ; Quaternary Ammonium Compounds ; Signal Transduction

Hepatocellular carcinoma (HCC) related to hepatitis B virus (HBV) and hepatitis C virus (HCV) infections is thought to account for more than 80% of primary liver cancers. Both HBV and HCV can establish chronic liver inflammatory infections, altering hepatocyte and liver physiology with potential liver disease progression and HCC development. Cyclophilin A (CypA) has been identified as an essential host factor for the HCV replication by physically interacting with the HCV non structural protein NS5A that in turn interacts with RNA-dependent RNA polymerase NS5B. CypA, a cytosolic binding protein of the immunosuppressive drug cyclosporine A, is overexpressed in many cancer types and often associated with malignant transformation. Therefore, CypA can be a good target for molecular cancer therapy. Because of antiviral activity, the CypA inhibitors have been tested for the treatment of chronic hepatitis C. Nonimmunosuppressive Cyp inhibitors such as NIM811, SCY-635, and Alisporivir have attracted more interests for appropriating CypA for antiviral chemotherapeutic target on HCV infection. This review describes CypA inhibitors as a potential HCC treatment tool that is contrived by their obstructing chronic HCV infection and summarizes roles of CypA in cancer development.
Carcinoma, Hepatocellular* ; Carrier Proteins ; Cyclophilin A* ; Cyclophilins ; Cyclosporine ; Cyclosporins ; Cytosol ; Hepacivirus* ; Hepatitis B virus ; Hepatitis C* ; Hepatitis C, Chronic ; Hepatitis ; Hepatocytes ; Liver ; Liver Diseases ; Liver Neoplasms ; RNA Replicase

Urolithiasis and calcium oxalate crystal deposition diseases are still significant medical problems. In the course of nephrocalcin cDNA cloning, we have identified FKBP-12 as an inhibitory molecule of calcium oxalate crystal growth. lambdagt 11 cDNA libraries were constructed from renal carcinoma tissues and screened for nephrocalcin cDNA clones using anti-nephrocalcin antibody as a probe. Clones expressing recombinant proteins, which appeared to be antigenically cross-reactive to nephrocalcin, were isolated and their DNA sequences and inhibitory activities on the calcium oxalate crystal growth were determined. One of the clone lambdagt 11 #31-1 had a partial fragment (80 bp) of FKBP-12 cDNA as an insert. Therefore, a full-length FKBP-12 cDNA was PCR-cloned from the lambdagt 11 renal carcinoma cDNA library and was subcloned into an expression vector. The resultant recombinant FKBP-12 exhibited an inhibitory activity on the calcium oxalate crystal growth (Kd=10(-7) M). Physiological effect of the extracellular FKBP-12 was investigated in terms of macrophage activation and proinflammatory cytokine gene induction. Extracellular FKBP-12 failed to activate macrophages even at high concentrations. FKBP-12 seems an anti-stone molecule for the oxalate crystal deposition disease and recurrent stone diseases.
Animals ; Base Sequence ; Calcium Oxalate/*antagonists & inhibitors ; Carcinoma, Renal Cell ; Crystallization ; DNA, Complementary ; Extracellular Space ; Glycoproteins/genetics ; Humans ; Kidney Calculi/*prevention & control ; Kidney Neoplasms ; Male ; Mice ; Mice, Inbred ICR ; Molecular Sequence Data ; Recombinant Fusion Proteins/genetics/metabolism ; Tacrolimus Binding Protein 1A/genetics/*metabolism

Animals ; Cyclophilins ; metabolism ; Liver ; metabolism ; pathology ; Liver Cirrhosis, Experimental ; metabolism ; Rats ; Rats, Wistar

To review B-cell activating factor (BAFF)-antagonist therapy in systemic lupus erythematosus (SLE), literature was searched using the search words and phrases, “BAFF”, “B lymphocyte stimulator (BLyS)”, “a proliferation-inducing ligand (APRIL)”, “B-cell maturation antigen (BCMA)”, “transmembrane activator and calcium-modulating and cyclophilin ligand interactor (TACI)”, “BLyS receptor 3 (BR3)”, “belimumab”, “atacicept”, “blisibimod”, “tabalumab”, and “lupus clinical trial”. In addition, papers from the author's personal library were searched. BAFF-antagonist therapy in SLE has a checkered past, with four late-stage clinical trials meeting their primary endpoints and four failing to do so. Additional late-stage clinical trials are enrolling subjects to address some of the remaining unresolved questions, and novel approaches are proposed to improve results. The BAFF-centric pathway is a proven therapeutic target in SLE. As the only pathway in the past 50+ years to have yielded an United States Food and Drug Administration-approved drug for SLE, it occupies a unique place in the armamentarium of the practicing rheumatologist. The challenges facing clinicians and investigators are how to better tweak the BAFF-centric pathway and improve on the successes realized.
B-Cell Activating Factor* ; B-Lymphocytes* ; Cyclophilins ; Humans ; Lupus Erythematosus, Systemic* ; Lymphocytes ; Research Personnel ; United States

To review B-cell activating factor (BAFF)-antagonist therapy in systemic lupus erythematosus (SLE), literature was searched using the search words and phrases, “BAFF”, “B lymphocyte stimulator (BLyS)”, “a proliferation-inducing ligand (APRIL)”, “B-cell maturation antigen (BCMA)”, “transmembrane activator and calcium-modulating and cyclophilin ligand interactor (TACI)”, “BLyS receptor 3 (BR3)”, “belimumab”, “atacicept”, “blisibimod”, “tabalumab”, and “lupus clinical trial”. In addition, papers from the author's personal library were searched. BAFF-antagonist therapy in SLE has a checkered past, with four late-stage clinical trials meeting their primary endpoints and four failing to do so. Additional late-stage clinical trials are enrolling subjects to address some of the remaining unresolved questions, and novel approaches are proposed to improve results. The BAFF-centric pathway is a proven therapeutic target in SLE. As the only pathway in the past 50+ years to have yielded an United States Food and Drug Administration-approved drug for SLE, it occupies a unique place in the armamentarium of the practicing rheumatologist. The challenges facing clinicians and investigators are how to better tweak the BAFF-centric pathway and improve on the successes realized.
B-Cell Activating Factor* ; B-Lymphocytes* ; Cyclophilins ; Humans ; Lupus Erythematosus, Systemic* ; Lymphocytes ; Research Personnel ; United States