CONSTRUCTION OF NERVE GROWTH FACTOR LOOP 4CONTAINING POLYPEPTIDES FOR FACILITATED GENE TRANSFER TO NEURONS JIEMING ZENG NATIONAL UNIVERISTY OF SINGAPORE 2004 Construction of Nerve Growth Factor Loop 4-Containing Polypeptides for Facilitated Gene Transfer to Neurons Jieming Zeng (MSc, MB) A THESIS SUMMITED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY FACULTY OF MEDICINE NATIONAL UNIVERISTY OF SINGAPORE & INSTITUTE OF BIOENGINEERING AND NANOTECHNOLOGY March 2004 ACKNOWLEDGEMENTS First and foremost, I wish to express my appreciation to my supervisor, A/P Shu Wang for his totally supporting on this study and for truly understanding what this research is all about And to A/P Heng-Phon Too and A/P Hanry Yu, my co-supervisors for the in-depth discussions and useful suggestions I would also like to acknowledge our exceptional research group at Institute of Bioengineering and Nanotechnology for providing such a fabulous environment for the study Especially thank Mr Shujun Gao for the assistance in animal studies, and thank Dr Xu Wang for the technical support in immunostaining study and confocal microscopy My thanks also to Ms Yuexia Ma for preparing the primary culture My gratitude also to Dr Alonzo H Ross from the University of Massachusetts Medical School for kindly providing two TrkA-expressing NIH3T3 cell lines Finally, I would like to express my gratitude to my family for their generosity, faith, and superb guidance during the lengthy PhD study To my father, Yaoying Zeng -immunologist and researcher -for rendering inspiring ideas To my mother, Xiaochang Cai -dermatologist and nurturer -for the continuous encouragement And my wife, Ruijuan Du who herself has been pursuing a PhD in molecular microbiology during the same period for believing in me from the start and lightening my life II TABLE OF CONTENTS Contents Page Acknowledgements………… ……… ………….………II Table of Contents……………………… ……….……… III List of Figures…………………………………………… VII Abbreviations………………………….… …………… VIII List of Publications and Patent………….………… … X Summary……………………… ……….…………………XII Introduction………….………… …………………… 1.1 Gene Therapy………………… ….…………………2 1.1.1 Background of Gene Therapy……… … …………….………2 1.1.2 Gene Delivery with Nonviral Vectors…………… …………….6 1.1.2.1 The importance of gene delivery vectors……………………6 1.1.2.2 The viral vectors…….………………………………………….7 1.1.2.3 The nonviral vectors………….………………………………10 1.1.2.4 The barriers to nonviral gene delivery….……… …………11 1.1.2.5 The improvement of nonviral vectors.……….… … …… 24 1.1.3 Targeted Gene Therapy…………………………………………25 1.1.3.1.Targeted gene therapy… ………………………………… 25 1.1.3.2 Approaches to targeted gene therapy.…… …………… 27 1.1.3.3 Targeting of nonviral vectors……………………………… 29 1.2 Gene Therapy in the Nervous System….… ….30 1.2.1 Gene Therapy in the Nervous System………… …… …… 30 1.2.1.1 The appeal to gene therapy in the nervous system… .30 III 1.2.1.2 The applicability of gene therapy in the nervous systems.….33 1.2.2 Targeted Gene Delivery to the Nervous System ………… 35 1.2.2.1 The challenges and requirements for gene therapy in the nervous system……………………………………… 35 1.2.2.2 Targeting of nonviral vectors to the nervous system… 37 1.2.3 NGF and NGF Peptidomimetics.……….………………………39 1.2.3.1 NGF and its receptors.….…….… …………………………39 1.2.3.2 NGF peptidomimetics… …………….……………… … 43 1.2.3.3 Targeting NGF receptor expressing neurons.…………….47 1.3 Aim of the Study……………… ………………… 48 Materials and Methods………… …………… …….50 2.1 Studies Using Bacterially Produced Polypeptides…………………………………………51 2.1.1 Plasmid Construction.……… ………………………….………51 2.1.2 Polypeptide Expression, Purification and Detection.……… 51 2.1.3 Cell Lines and Reporter Plasmid………… ………………….52 2.1.4 Detection of TrkA, Erk and Akt Activation.… ……………….53 2.1.5 Cell Survival Assay.…………………………… …………… 54 2.1.6 DNA Retardation Assay……………………… ………………54 2.1.7 Gene Delivery Assay….…………………… …………………55 2.2 Studies Using Chemically Synthesized Peptides……………….………………………… 56 2.2.1 Peptide Design and Synthesis….……………… …… …… 56 2.2.2 Cell Cultures……………………………………… ……………56 2.2.3 Biochemical and Biological Assays…………… …………….58 IV 2.2.4 Report Plasmid, DNA Binding Assay and Preparation of DNA complexes……………….……………… 60 2.2.5 Zeta Potential and Size of the Complexes………… ……….61 2.2.6 Gene Transfer.……………………………………… ……… 62 2.2.7 Flow Cytometry, Immunocytochemistry and Immunohistochemistry…………………………………………… 63 Experimental Results………………………… ………66 3.1 Studies Using Bacterially Produced Polypeptides…………………………………………67 3.1.1 Description of the Recombinant Cationic Polypeptides….… 67 3.1.2 Activation of TrkA, Erk and Akt by DsbC-NL4-10K………… 70 3.1.3 Promotion of PC12 Cell Survival by DsbC-NL4-10K ……….72 3.1.4 Binding of DsbC-NL4-10K to Plasmid DNA… …………… 74 3.1.5 Enhanced Polycation-mediated Gene Delivery to PC12 by DsbC-NL4-10K……………… …………………… 76 3.2 Studies Using Chemically Synthesized Peptides……………….………………………… 81 3.2.1 Biochemical and Biological Effects of NL4-10K Peptide………………………………………………………… 81 3.2.2 NL4-10K Binds to DNA and Mediates In Vitro Gene Transfer… ……………………………………………… 86 3.2.3 NL4-10K Mediates Gene Delivery through TrkA…………… 90 3.2.4 NL4-10K-containing Complexes Mediate Gene Delivery to DRG In Vivo…….……………………………… …100 3.2.5 Biocompatibility of PEI600/DNA/NL4-10K Ternary Complexes ………………………………………… 104 V Discussion……………………………………… …….107 4.1 Studies Using Bacterially Produced Polypeptides……………………………………… 108 4.1.1 The design and production of polypeptides………………….108 4.1.2 The polypeptide-containing complexes… ………………….111 4.2 Studies Using Chemically Synthesized Peptides……………….………………… …… 112 4.2.1 The design of targeting ligand….…………………………… 113 4.2.2 The binding of ligand to TrkA………………………………….114 4.2.3 The selectivity in peptide-mediated gene delivery………… 115 4.2.4 The peptide-containing complexes ….……… …………….117 4.2.5 The use of oligolysine as DNA carrier…….………………….118 4.2.6 The possible intrinsic gene delivery at high +/- charge ratio………………………………………….………119 4.2.7 The competitive inhibition assay…………………………… 122 4.2.8 The possibility of receptor-mediated gene delivery using a targeted oligolysine-based system at high +/- charge ratio…………………………….……………………124 4.2.9 The application………………………………………………….125 References…………………………………………… 127 Appendix A: Amino Acid Sequences……… ………146 Appendix B: Nucleic Acid Sequences……………….148 VI LIST OF FIGURES Figures Page Figure 3.1 Schematic of expression plasmids… …………………………….68 Figure 3.2 Structure and production of recombinant polypeptides …………69 Figure 3.3 Activation of TrkA, Erk and Akt by DsbC-NL4-10K……………….71 Figure 3.4 Promotion of neuronally differentiated PC12 cell survival in serum-free medium by DsbC-NL4-10K…………………… ………………73 Figure 3.5 DNA retardation by DsbC-NL4-10K…………………………… …75 Figure 3.6 Enhanced PEI600-mediated gene transfer by DsbC-NL4-10K in PC12 cells…… …………………………………………… 78 Figure 3.7 Comparison of DsbC-NL4-10K-meidated gene delivery in PC12 and COS7 cells…………….…………………………………………….79 Figure 3.8 Competitive inhibition of DsbC-NL4-10K-mediated gene delivery to PC12 cells by DsbC-NL4-10K pre-treatment……………….…… 80 Figure 3.9 Structures of chimeric peptide NL4-10K and its control NL4…….83 Figure 3.10 Effects of NL4-10K on TrkA receptor…………………………… 84 Figure 3.11 Promotion of neuronally differentiated PC12 cell survival in serum-free medium by NL4-10K….…………………………………85 Figure 3.12 DNA retardation by NL4-10K in agarose gel under electrophoresis……… …………………………………………………88 Figure 3.13 Efficiency of NL4-10K-mediated gene delivery in vitro………….89 Figure 3.14 Flow cytometric analysis of Trk receptors in various cell lines and primary cultured cells…………………………………………… 91 Figure 3.15 Specificity of NL4-10K-mediated gene delivery………………….97 Figure 3.16 Co-localization of Trk receptors and luciferase immunoreactivity in primary cortical neurons after transfection with NL4-10K/pCAGluc complexes……………………………………… …….99 Figure 3.17 Gene delivery mediated by 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KKKKKKK 50 100 150 200 250 Features: - aa 1-236: DsbC - aa 244-249: His6 tag - aa 259-287: NL4 Name: DsbC-NL4-10K Sequence: MKKGFMLFTL LTNSGVLYIT YKAPQEKHVI DAEKEMKAIW GTPAVVLSNG AGLVPRGSCT LAAFSGFAQA DDGKHIIQGP TVFTDITCGY CAKDKNKAFD TLVPGYQPPK TTHTFVKALT DDAAIQQTLA MYDVSGTAPV CHKLHEQMAD DVMAGKSVAP EMKEFLDEHQ MDGKQAAWRF Features: - aa 1-236: DsbC - aa 244-249: His6 tag - aa 259-297: NL4-10K Name: NL4 Sequence: CTTTHTFVKA LTMDGKQAAW RFIRIDTAC Features: - aa 1-29 correspond to aa 80-108 of nerve growth factor (NGF) and include loop 4, a known receptor binding region - C1 and C29 form a disulfide bridge Name: NL4-10K Sequence: CTTTHTFVKA LTMDGKQAAW RFIRIDTACK KKKKKKKKK Features: - aa 1-29 correspond to aa 80-108 of nerve growth factor (NGF) and include loop 4, a known receptor binding region 146 - C1 and C29 form a disulfide bridge aa 30-39 form a nucleic acid binding domain Name: 10K Sequence: KKKKKKKKKK 147 APPENDIX B: NUCLEIC ACID SEQUENCES NA SEQ Description: Forward primer for NL4 and NL4-10K Sequence: 5’-TGTACCACGA CTCACACC-3’ NA SEQ Description: Reverse primer for NL4 Sequence: 5’-GCAAGCTTTC AACAGGCCGT ATCTATCCG-3’ NA SEQ Description: Reverse primer for NL4-10K Sequence: 5’-GCAAGCTTTC ATTTTTTTTT TTTTTTTTTT TTTTTTTTTT TACAGGCCGT ATCTATCCG-3’ NA SEQ Description: Coding sequence for Sequence: atgaagaaag gttttatgtt gtttactttg tgctcaggct gatgacgcgg caattcaaca tcaaaagcag cgatattcag cccgcgcctg ctgactaaca gcggcgtgtt gtacatcacc tcaggggcca atgtatgacg ttagtggcac ataagatgct gttaaagcag ttgaatgcgc tataaagcgc cgcaggaaaa acacgtcatc ctgtggttac tgccacaaac tgcatgagca tggggatcac cgtgcgttat cttgctttcc gatgcagaga aagaaatgaa agctatctgg agcgtttgat gatgtgatgg caggtaaaag acgtggatat tgccgaccat tacgcacttg ggtactccgg cagttgtgct gagcaatggc gccgccgaaa gagatgaaag aatttctcga gcggtaaagg atcaactagt ggttctggtc gcgggtctgg tgccacgcgg tagttgtacc ggcgctgacc atggatggca agcaggctgc atacggcctg t DsbC-NL4 ttagcggcgt aacgttagcc tagctggcat gatgatggta ggctccggtc ttgaaaaaga accgtgttta aatggcagac cgcgccaggg tgtgcgaaag cgtcgcacca gcgtccagct acacttgttc cgaacaccaa atcaccatca acgactcaca ctggcggttt tttcaggctt aaaatgggca gaagacagtt aacatatcat aatgtcacca gatgatcgtt ctgatattac tacaacgcgc gctggacagc ataaaaacaa gccagttgcg tggcgttagc cgggttacca aaaatgacca ccatcactcc cctttgtcaa atccggatag 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 NA SEQ Description: Coding sequence for Sequence: atgaagaaag gttttatgtt gtttactttg tgctcaggct gatgacgcgg caattcaaca tcaaaagcag cgatattcag cccgcgcctg ctgactaaca gcggcgtgtt gtacatcacc tcaggggcca atgtatgacg ttagtggcac ataagatgct gttaaagcag ttgaatgcgc tataaagcgc cgcaggaaaa acacgtcatc ctgtggttac tgccacaaac tgcatgagca DsbC-NL4-10K ttagcggcgt aacgttagcc tagctggcat gatgatggta ggctccggtc ttgaaaaaga accgtgttta aatggcagac tttcaggctt aaaatgggca gaagacagtt aacatatcat aatgtcacca gatgatcgtt ctgatattac tacaacgcgc 50 100 150 200 250 300 350 400 148 tggggatcac gatgcagaga agcgtttgat acgtggatat ggtactccgg gccgccgaaa gcggtaaagg gcgggtctgg ggcgctgacc atacggcctg cgtgcgttat aagaaatgaa gatgtgatgg tgccgaccat cagttgtgct gagatgaaag atcaactagt tgccacgcgg atggatggca taaaaaaaaa cttgctttcc agctatctgg caggtaaaag tacgcacttg gagcaatggc aatttctcga ggttctggtc tagttgtacc agcaggctgc aaaaaaaaaa cgcgccaggg tgtgcgaaag cgtcgcacca gcgtccagct acacttgttc cgaacaccaa atcaccatca acgactcaca ctggcggttt aaaaaaaaaa gctggacagc ataaaaacaa gccagttgcg tggcgttagc cgggttacca aaaatgacca ccatcactcc cctttgtcaa atccggatag 450 500 550 600 650 700 750 800 850 149 .. .Construction of Nerve Growth Factor Loop 4- Containing Polypeptides for Facilitated Gene Transfer to Neurons Jieming Zeng (MSc, MB) A THESIS SUMMITED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY... success of gene therapy for neurological disorders In this study, chimeric polypeptides were constructed for targeted gene transfer to cells expressing nerve growth factor (NGF) receptor TrkA... Targeting of nonviral vectors As there is no panacea, there is no versatile vector for gene therapy to all kind of diseases Gene delivery vector has to be customized to fit the requirement of various