Fisher John P. e.a. (ed.) Tissue Engineering

Подождите немного. Документ загружается.

mikos: “9026_c013” — 2007/4/9 — 15:51 — page 20 — #20

13-20 Tissue Engineering

[114] Zalipsky, S., Chemistry of polyethylene glycol conjugates with biologically active molecules, Adv.

Drug Deliv. Rev., 16, 157, 1995.

[115] Veronese, F.M. and Harris, J.M., Introduction and overview of peptide and protein pegylation,

Adv. Drug Deliv. Rev., 54, 453, 2002.

[116] Zhdanov, V.P. and Kasemo, B., Monte Carlo simulations of the kinetics of protein adsorption,

Surf.Rev.Lett., 5, 615, 1998.

[117] Norde, W., Adsorption of proteins from solution at the solid–liquid interface, Adv. Colloid Interface

Sci., 25, 267, 1986.

[118] Malmsten, M., Protein adsorption at the solid–liquid interface, in Protein Architecture,Lvov,Y.

and Moehwald, H., Eds., New York, Marcel Dekker, 1–23, 2000.

[119] Felgner, P.L. and Wilson, J.E., Hexokinase binding to polypropylene test tubes. Artifactual activity

losses from protein binding to disposable plastics, Anal. Biochem., 74, 631, 1976.

[120] Beyerman, H.C. et al., On the instability of secretin, Life Sci., 29, 885, 1981.

[121] Kramer, K.J. et al., Puriﬁcation and characterization of the carrier protein for juvenile hor-

mone from the hemolymph of the tobacco hornworm Manduca sexta Johannson (Lepidoptera:

Sphingidae), J. Biol. Chem., 251, 4979, 1976.

[122] Suelter, C.H. and DeLuca, M., How to prevent losses of protein by adsorption to glass and plastic,

Anal. Biochem., 135, 112, 1983.

[123] Katakam, M. and Banga, A.K., Use of poloxamer polymers to stabilize recombinant human growth

hormone against various processing stresses, Pharm. Dev. Technol., 2, 143, 1997.

[124] Charman, S.A., Mason, K.L., and Charman, W.N., Techniques for assesing the effects of phar-

maceutical excipients on the aggregation of porcine growth hormone, Pharm. Res., 10, 954,

1993.

[125] Oliva, A. et al., Effect of high shear rate on stability of proteins: kinetic study, J. Pharm. Biomed.

Anal., 33, 145, 2003.

[126] Spenlehauer, G., Spenlehauer-Bonthonneau, F., and Thies, C., Biodegradable microparticles for

delivery of polypeptides and proteins, Prog. Clin. Biol. Res., 292, 283, 1989.

[127] van Erp, S.H., Kamenskaya, E.O., and Khmelnitsky, Y.L., The effect of water content and nature

of organic solvent on enzyme activity in low-water media. A quantitative description, Eur. J.

Biochem./FEBS, 202, 379, 1991.

[128] Khmelnitsky, Y.L. et al., Denaturation capacity: a new quantitative criterion for selection of organic

solvents as reaction media in biocatalysis, Eur. J. Biochem./FEBS, 198, 31, 1991.

[129] Brunner, A., Mader, K., and Gopferich, A., The chemical microenvironment inside biodegradable

microspheres during erosion, Proceedings of the International Symposium on Controlled Release of

Bioactive Materials, Vol. 25, p. 154, 1998.

[130] Lucke, A. et al., The effect of poly(ethylene glycol)–poly(d,l-lactic acid) diblock copolymers on

peptide acylation, J. Control. Release, 80, 157, 2002.

[131] Lucke, A., Kiermaier, J., and Gopferich, A., Peptide acylation by poly(.alpha.-hydroxy esters),

Pharm. Res., 19, 175, 2002.

[132] Schwendeman, S.P., Recent advances in the stabilization of proteins encapsulated in injectable

PLGA delivery systems, Crit. Rev. Ther. Drug Carrier Syst., 19, 73, 2002.

[133] Zhu, G., Mallery, S.R., and Schwendeman, S.P., Stabilization of proteins encapsulated in injectable

poly(lactide-co-glycolide), Nat. Biotechnol., 18, 52, 2000.

[134] Lam, X.M., Duenas, E.T., and Cleland, J.L., Encapsulation and stabilization of nerve growth factor

into poly(lactic-co-glycolic) acid microspheres, J. Pharm. Sci., 90, 1356, 2001.

[135] Pean, J.M. et al., Why does PEG 400 co-encapsulation improve NGF stability and release from

PLGA biodegradable microspheres? Pharm. Res., 16, 1294, 1999.

[136] Tang, B., Wang, M., and Wise, C., Nerve growth factor mRNA stability is controlled by a cis-acting

instability determinant in the 3



-untranslated region, Mol. Brain Res., 46, 118, 1997.

[137] Shaw, G. and Kamen, R., A conserved AU sequence from the 3



untranslated region of GM-CSF

mRNA mediates selective mRNA degradation, Cell, 46, 659, 1986.

mikos: “9026_c013” — 2007/4/9 — 15:51 — page 21 — #21

Drug Delivery 13-21

[138] Schiavi, S.C., Belasco, J.G., and Greenberg, M.E., Regulation of proto-oncogene mRNA stability,

Biochim. Biophys. Acta, 1114, 95, 1992.

[139] Nimni, M.E., Polypeptide growth factors: targeted delivery systems, Biomaterials, 18, 1201, 1997.

[140] Davis, G.E. and Camarillo, C.W., Regulation of endothelial cell morphogenesis by integrins,

mechanical forces, and matrix guidance pathways, Exp. Cell Res., 216, 113, 1995.

[141] Lee, K.Y. et al., Controlled growth factor release from synthetic extracellular matrices, Nature, 408,

998, 2000.

[142] Wallace, D.G. and Rosenblatt, J., Collagen gel systems for sustained delivery and tissue engineering,

Adv. Drug Deliv. Rev., 55, 1631, 2003.

[143] Drury, J.L. and Mooney, D.J., Hydrogels for tissue engineering: scaffold design variables and

applications, Biomaterials, 24, 4337, 2003.

[144] Peters, M.C. et al., Release from alginate enhances the biological activity of vascular endothelial

growth factor, J. Biomater. Sci., Polym. Ed., 9, 1267, 1998.

[145] Taipale, J. and Keski-Oja, J., Growth factors in the extracellular matrix, FASEB J., 11, 51, 1997.

[146] Langer, R. and Tirrell, D.A., Designing materials for biology and medicine, Nature (London, United

Kingdom), 428, 487, 2004.

[147] Majeti, N.V. and Ravi Kumar, M.N.V., Nano and microparticles as controlled drug delivery devices,

J. Pharm. Pharm. Sci. [online computer ﬁle], 3, 234, 2000.

[148] Lopez, V.C. and Snowden, M.J., The role of colloidal microgels in drug delivery, Drug Deliv. Syst.

Sci., 3, 19, 2003.

[149] Saltzman, W.M., Delivering tissue regeneration, Nat. Biotechnol., 17, 534, 1999.

[150] Xu, R. et al., Diabetes gene therapy: potential and challenges, Curr.GeneTher., 3, 65, 2003.

[151] Merdan, T., Kopecek, J., and Kissel, T., Prospects for cationic polymers in gene and oligonucleotide

therapy against cancer, Adv. Drug Deliv. Rev., 54, 715, 2002.

[152] Lollo, C.P., Banaszczyk, M.G., and Chiou, H.C., Obstacles and advances in non-viral gene delivery,

Curr. Opin. Mol. Ther., 2, 136, 2000.

[153] Johnson-Saliba, M. and Jans, D.A., Gene therapy: optimising DNA delivery to the nucleus, Curr.

Drug Targets, 2, 371, 2001.

[154] Bout, A. and Crucell, L., Gene therapy, in Pharmaceutical Biotechnology, 2nd ed., Commelin,

D.J.A. and Sindelar, R.D., Eds., London, Taylor & Francis Ltd., 175–192, 2002.

[155] Kuhl, P.R. and Grifﬁth-Cima, L.G., Tethered epidermal growth factor as a paradigm for growth

factor-induced stimulation from the solid phase, Nat. Med., 2, 1022, 1996.

[156] Blunk, T., Goepferich, A., and Tessmar, J., Special issue biomimetic polymers, Biomaterials, 24,

4335, 2003.

[157] Aldersey-Williams, H., Towards biomimetic architecture, Nat. Mater., 3, 277, 2004.

[158] Ito, Y., Tissue engineering by immobilized growth factors, Mater. Sci. Eng. C: Biomimetic Mater.,

Sensors Syst., C6, 267, 1998.

[159] Shakesheff, K.M., Cannizzaro, S.M., and Langer, R., Creating biomimetic micro-environments

with synthetic polymer–peptide hybrid molecules, J. Biomater. Sci., Polym. Ed., 9, 507, 1998.

[160] Thayumanavan, S. et al., Towards dendrimers as biomimetic macromolecules, C. R. Chimie,6,

767, 2003.

[161] Hoffman, A.S. et al., Design of “smart” polymers that can direct intracellular drug delivery, Polym.

Adv. Technol., 13, 992, 2002.

[162] Peppas, N.A. and Huang, Y., Polymers and gels as molecular recognition agents, Pharm. Res., 19,

578, 2002.

[163] Langer, R. and Folkman, J., Polymers for the sustained release of proteins and other

macromolecules, Nature, 263, 797.

[164] Saltzman, W.M. and Langer, R., Transport rates of proteins in porous materials with known

microgeometry, Biophys. J., 55, 163, 1989.

[165] Bawa, R. et al., An explanation for the controlled release of macromolecules from polymers,

J. Control. Release, 1, 259, 1985.

mikos: “9026_c013” — 2007/4/9 — 15:51 — page 22 — #22

13-22 Tissue Engineering

[166] Leong, K.W. et al., Polyanhydrides for controlled release of bioactive agents, Biomaterials, 7, 364,

1986.

[167] Heller, J., Controlled drug release from poly(ortho esters)—asurfaceeroding polymer, J. Control.

Release, 2, 167, 1985.

[168] Goepferich, A., Mechanisms of polymer degradation and erosion, Biomaterials, 17, 103, 1996.

[169] Goepferich, A., Polymer degradation and erosion. Mechanisms and applications, Eur. J. Pharm.

Biopharm., 42, 1, 1996.

[170] Fu, K. et al., Visual evidence of acidic environment within degrading poly(lactic-co-glycolic acid)

(PLGA) microspheres, Pharm. Res., 17, 100, 2000.

[171] Brunner, A., Mader, K., and Gopferich, A., pH and osmotic pressure inside biodegradable

microspheres during erosion, Pharm. Res., 16, 847, 1999.

[172] Lucke, A., Kiermaier, J., and Gopferich, A., Peptide acylation by poly(a-hydroxy esters), Pharm.

Res., 19, 175, 2002.

[173] Athanasiou, K.A. et al., Orthopaedic applications for PLA–PGA biodegradable polymers, Arth-

roscopy: The Journal of Arthroscopic and Related Surgery: Ofﬁcial Publication of the Arthroscopy

Association of North America and the International Arthroscopy Association, 14, 726, 1998.

[174] Biggs, D.L. et al., In vitro and in vivo evaluation of the effects of PLA microparticle crystallinity on

cellular response, J. Control. Release: Ofﬁcial Journal of the Controlled Release Society, 92, 147, 2003.

[175] Sandor, M. et al., Effect of protein molecular weight on release from micron-sized PLGA

microspheres, J. Control. Release, 76, 297, 2001.

[176] Tabata, Y. and Ikada, Y., Protein release from gelatin matrixes, Adv. Drug Deliv. Rev., 31, 287, 1998.

[177] Gombotz, W.R. and Wee, S., Protein release from alginate matrixes, Adv. Drug Deliv. Rev., 31, 267,

1998.

[178] Fujioka, K. et al., Protein release from collagen matrixes, Adv. Drug Deliv. Rev., 31, 247, 1998.

[179] Sinha, V.R. and Trehan, A., Biodegradable microspheres for protein delivery, J. Control. Release,

90, 261, 2003.

[180] Sinha, V.R. et al., Poly-e-caprolactone microspheres and nanospheres: an overview, Int. J. Pharm.,

278, 1, 2004.

[181] Orive, G. et al., Cell microencapsulation technology for biomedical purposes: novel insights and

challenges, Trends Pharmacol. Sci., 24, 207, 2003.

[182] Orive, G., Hernandez, R.M., Gascón, A.R., and Pedraz, J.L., Challenges in cell encapsulation, in

Cell Immobilization Biotechnology. Part II. Applications,Nedoviˇc, N. and Willaert, R., Eds., “Focus

on Biotechnology” series, Dordrecht, The Netherlands, Kluwer, 9999, 1991.

[183] Giannoni, P. and Hunziker, E.B., Release kinetics of transforming growth factor beta1 from ﬁbrin

clots, Biotechnol. Bioeng., 83, 121, 2003.

[184] Collier, J.H. and Messersmith, P.B., Phospholipid strategies in biomineralization and biomaterials

research, Ann. Rev. Mater. Res., 31, 237, 2001.

[185] Haller, M.F. and Saltzman, W.M., Nerve growth factor delivery systems, J. Control. Release, 53, 1,

1998.

[186] Saltzman, W.M. et al., Intracranial delivery of recombinant nerve growth factor: release kinetics

and protein distribution for three delivery systems, Pharm. Res., 16, 232, 1999.

[187] Lee, J.E. et al., Effects of the controlled-released TGF-b1 from chitosan microspheres on chon-

drocytes cultured in a collagen/chitosan/glycosaminoglycan scaffold, Biomaterials, 25, 4163,

2004.

[188] Cleland, J.L. et al., Recombinant human growth hormone poly(lactic-glycolic acid) (PLGA)

microspheres provide a long lasting effect, J. Control. Release, 49, 193, 1997.

[189] Cleland, J.L., Protein delivery from biodegradable microspheres, Pharm. Biotechnol., 10, 1, 1997.

[190] Van de Weert, M., Hennink, W.E., and Jiskoot, W., Protein instability in poly(lactic-co-glycolic

acid) microparticles, Pharm. Res., 17, 1159, 2000.

[191] Tonnesen, H.H. and Karlsen, J., Alginate in drug delivery systems, Drug Dev. Ind. Pharm.,

28, 621, 2002.

mikos: “9026_c013” — 2007/4/9 — 15:51 — page 23 — #23

Drug Delivery 13-23

[192] Gu, F., Amsden, B., and Neufeld, R., Sustained delivery of vascular endothelial growth factor with

alginate beads, J. Control. Release, 96, 463, 2004.

[193] Mladenovska, K. et al., BSA-loaded gelatin microspheres: preparation and drug release rate in the

presence of collagenase, Acta Pharm. (Zagreb, Croatia), 52, 91, 2002.

[194] Tabata, Y. et al., De novo formation of adipose tissue by controlled release of basic ﬁbroblast growth

factor, Tissue Eng., 6, 279, 2000.

[195] Bummer, P.M., Physical chemical considerations of lipid-based oral drug delivery — solid lipid

nanoparticles, Crit. Rev. Ther. Drug Carrier Syst., 21, 1, 2004.

[196] Maschke, A. et al., Lipids: an alternative material for protein and peptide release, ACS Symp. Ser.,

879, 176, 2004.

[197] Cortesi, R. et al., Production of lipospheres as carriers for bioactive compounds, Biomaterials, 23,

2283, 2002.

[198] Reithmeier, H., Herrmann, J., and Gopferich, A., Lipid microparticles as parenteral controlled

release device for peptides, J. Control. Release, 73, 339, 2001.

[199] Reithmeier, H., Gopferich, A., and Herrmann, J., Preparation and characterization of lipid micro-

particles containing thymocartin, an immunomodulating peptide, Proceedings of the International

Symposium on Controlled Release of Bioactive Materials, Vol. 26, p. 681, 1999.

[200] Lee, K.Y. and Mooney, D.J., Hydrogels for tissue engineering, Chem. Rev., 101, 1869, 2001.

[201] Yamamoto, M., Takahashi, Y., and Tabata, Y., Controlled release by biodegradable hydrogels

enhances the ectopic bone formation of bone morphogenetic protein, Biomaterials, 24, 4375,

2003.

[202] Yamamoto, M. et al., Bone regeneration by transforming growth factor b1 released from a

biodegradable hydrogel, J. Control. Release, 64, 133, 2000.

[203] Tabata, Y., Matsui, Y., and Ikada, Y., Growth factor release from amylopectin hydrogel based on

copper coordination, J. Control. Release, 56, 135, 1998.

[204] Zisch, A.H., Lutolf, M.P., and Hubbell, J.A., Biopolymeric delivery matrices for angiogenic growth

factors, Cardiovasc. Pathol., 12, 295, 2003.

[205] Jeong, B. et al., Biodegradable block copolymers as injectable drug-delivery systems, Nature, 388,

860, 1997.

[206] Ebara, M. et al., Temperature-responsive cell culture surfaces enable “On-Off” afﬁnity control

between cell integrins and RGDS ligands, Biomacromolecules, 5, 505, 2004.

[207] Zisch, A.H. et al., Cell-demanded release of VEGF from synthetic, biointeractive cell-ingrowth

matrices for vascularized tissue growth, FASEB J., 17, 2260, 2003.

[208] Sakiyama-Elbert, S.E. and Hubbell, J.A., Controlled release of nerve growth factor from a heparin-

containing ﬁbrin-based cell ingrowth matrix, J. Control. Release, 69, 149, 2000.

[209] Kimura, Y. et al., Adipose tissue engineering based on human preadipocytes combined

with gelatin microspheres containing basic ﬁbroblast growth factor, Biomaterials, 24, 2513,

2003.

[210] Holland, T.A., Tabata, Y., and Mikos, A.G., In vitro release of transforming growth factor-b1

from gelatin microparticles encapsulated in biodegradable, injectable oligo(poly(ethylene glycol)

fumarate) hydrogels, J. Control. Release, 91, 299, 2003.

[211] Holland, T.A. et al., Transforming growth factor-b1 release from oligo(poly(ethylene glycol)

fumarate) hydrogels in conditions that model the cartilage wound healing environment, J. Control.

Release, 94, 101, 2004.

[212] Hu, Y., Hollinger, J.O., and Marra, K.G., Controlled release from coated polymer microparticles

embedded in tissue-engineered scaffolds, J. Drug Target., 9, 431, 2001.

[213] Kim, H., Kim, H.W., and Suh, H., Sustained release of ascorbate-2-phosphate and dexa-

methasone from porous PLGA scaffolds for bone tissue engineering using mesenchymal stem

cells, Biomaterials, 24, 4671, 2003.

[214] Ziegler, J. et al., Adsorption and release properties of growth factors from biodegradable implants,

J. Biomed. Mater. Res., 59, 422, 2002.

mikos: “9026_c013” — 2007/4/9 — 15:51 — page 24 — #24

13-24 Tissue Engineering

[215] Tabata, Y. and Ikada, Y., Vascularization effect of basic ﬁbroblast growth factor released from

gelatin hydrogels with different biodegradabilities, Biomaterials, 20, 2169, 1999.

[216] Ueda, H. et al., Use of collagen sponge incorporating transforming growth factor-beta1 to promote

bone repair in skull defects in rabbits, Biomaterials, 23, 1003, 2002.

[217] Whang, K., Goldstick, T.K., and Healy, K.E., A biodegradable polymer scaffold for delivery of

osteotropic factors, Biomaterials, 21, 2545, 2000.

[218] Whang, K. et al., Ectopic bone formation via rhBMP-2 delivery from porous bioabsorbable

polymer scaffolds, J. Biomed. Mater. Res., 42, 491, 1998.

[219] Jang, J.H. and Shea, L.D., Controllable delivery of non-viral DNA from porous scaffolds, J. Control.

Release, 86, 157, 2003.

[220] Brunner A., Gopferich, A., Characterization of Polyanhydride Microspheres, p. 169, 1996.

[221] Sohier, J. et al., A novel method to obtain protein release from porous polymer scaffolds: emulsion

coating, J. Control. Release, 87, 57, 2003.

[222] Li, X. et al., Inﬂuence of process parameters on the protein stability encapsulated in poly-dl-

lactide-poly(ethylene glycol) microspheres, J. Control. Release, 68, 41, 2000.

[223] Crotts, G. and Park, T.G., Protein delivery from poly(lactic-co-glycolic acid) biodegradable

microspheres: release kinetics and stability issues, J. Microencapsul., 15, 699, 1998.

[224] Jiang, G. et al., Assessment of protein release kinetics, stability and protein polymer interaction

of lysozyme encapsulated poly(d,l-lactide-co-glycolide) microspheres, J. Control. Release, 79, 137,

2002.

[225] Sirotkin, V.A. et al., Calorimetric and Fourier transform infrared spectroscopic study of solid

proteins immersed in low water organic solvents, Biochim. Biophys. Acta, 1547, 359, 2001.

[226] Shea, L.D. et al., DNA delivery from polymer matrices for tissue engineering, Nat. Biotechnol., 17,

551, 1999.

mikos: “9026_c014” — 2007/4/9 — 15:51 — page1—#1

Gene Therapy

J.M. Munson

W.T. Godbey

Tulane University

14.1 Introduction.............................................. 14-1

14.2 Nucleotides for Delivery................................. 14-2

DNA (deoxyribonucleic acid) • RNA

14.3 Gene Delivery ............................................ 14-2

Biological Delivery Methods • Chemical Delivery Methods

• Physical Delivery Methods

14.4 Intracellular Pathways ................................... 14-6

14.5 Cell and Tissue Targeting................................ 14-7

14.6 Applications .............................................. 14-8

In Vitro • Ex Vivo • In Vivo

14.7 Clinical Applications .................................... 14-9

14.8 Summary ................................................. 14-10

References ....................................................... 14-10

14.1 Introduction

Gene therapy is the delivery of genetic material into cells for the purpose of altering cellular function. This

seemingly straightforward deﬁnition encompasses a variety of situations that can at times seem unrelated.

The delivered genetic material can be composed of deoxyribonucleic acid (DNA) or RNA, or even involve

proteins in some cases. The alteration in cellular function can be an increase or decrease in the amount

of a native protein that is produced, or the production of a protein that is foreign. The delivery of the

genetic material can occur directly, as is the case with microinjection, or involve carriers that interact with

cell membranes or membrane-bound proteins as a part of cellular entry. Polynucleotides can be single or

double stranded, and can code for a message, or not (as is the case for antisense gene delivery). Even the

location of cells at the time of gene delivery is not restricted. Cells can be part of a living organism, can

exist as a culture on a plate, or can be removed from an organism, transfected, and replaced into the same

or a different organism at a later time.

Gene therapy came into being after the development of recombinant DNA technology and initially

moved forward using viruses to target sites in vitro [1]. With the understanding of retroviruses and

their possible uses as vectors for delivery, gene therapy progressed to applications involving mammalian

organisms during the 1980s [1]. Since then, new techniques for gene delivery have been developed

that utilize both viral and nonviral carriers. These techniques have been successful enough that proposed

disease treatments have evolved to the clinical trial stage with encouraging success. Although many cellular

processing mechanisms remain unclear and the search for the ideal gene delivery vector remains ongoing,

the tools and methods for gene therapy have provided a strong base from which to build.

14-1

mikos: “9026_c014” — 2007/4/9 — 15:51 — page2—#2

14-2 Tissue Engineering

14.2 Nucleotides for Delivery

14.2.1 DNA (deoxyribonucleic acid)

DNA is the building block of life and as such it remains a staple in the delivery of genetic material to the

cell. There are many strategies as to how the DNA will interact with the existing genome and what is the

best way to produce the desired effect.

14.2.1.1 Plasmids

A plasmid is a circular piece of DNA. Extrachromosomal plasmids can be replicated or transcribed inde-

pendently of the rest of the DNA in the nucleus. This attribute makes plasmids an ideal tool for gene

therapy. Plasmids can include a selectable marker for identiﬁcation of transfectants/transductants, a mul-

tiple cloning site for ease of inserting/deleting additional nucleotide strands, the exon(s) of interest, and

regulatory/binding elements such as promoters and enhancers. A common promoter used in gene therapy

is the cytomegalovirus immediate early promoter (CMV

), used because of its strength in transcription

initiation in nearly every mammalian cell. However, several other promoters have been investigated, such

as the glucose-related protein promoter [2] and many cell-speciﬁc promoters. Enhancers include the

simian virus 40 (SV

) enhancer as well as long terminal repeats (LTRs) [2]. The exons that are delivered

may code for necessary cell-speciﬁc proteins, engineered protein polymers [3], or what has become a

common objective in cancer cell research: suicide genes [4].

14.2.1.2 Nucleotide Decoys

DNA and RNA decoys are small oligonucleotide fragments that mimic the start sequences of potentially

harmful genes. By doing so, the decoys can effectively trap the transcriptional and translational machinery,

thereby causing a sharp decrease in mRNA or protein production. There has been success with this

approach in human immunodeﬁciency virus (HIV) research. DNA and RNA decoys have been used to

halt the function of REV proteins, which are responsible for making late transcripts of RNA and exporting

them to the cytoplasm [5], and the TAT protein, which binds and activates the natural promoter for

HIV-1 [6].

14.2.2 RNA

RNA also holds a valuable place in gene therapy because it can be used to alter cellular function. Two

examples of RNA use in gene therapy are antisense RNA and small interfering RNA (siRNA). Antis-

ense RNA provides functional alterations in cells by acting on host gene products post-transcriptionally.

Antisense RNA functions by binding to cellular mRNA transcripts, which prevents ribosomal binding

and therefore translation. Antisense RNAs have been investigated for their use in applications such as

beta-globin gene inhibition as a possible treatment for sickle cell anemia [7], bcr/abl interference in myel-

oid leukemia research [8], and CXCR4 disruption for HIV-1 gene therapy [9], among others. The use

of siRNA can interfere directly with genomic DNA transcription. First described by Elbashir et al. [10]

in 2001, siRNAs have very high speciﬁcity that can be used to target a single-nucleotide polymorphism

(SNP) on a single mutant allele [11,12]. The result would be to silence the mutant allele while permitting

continued transcription of the wild type gene. This technology has been used in vivo to successfully target

spinocerebellar ataxia type 3 and frontotemporal dementia [13].

14.3 Gene Delivery

Simply administering naked genetic material in the vicinity of cell exteriors is usually not sufﬁcient to

bring about the desired cellular response at adequate levels. Carriers have been employed to aid in the

transfer of genetic material from the cell exterior to the cytoplasm or nucleus. There is a wide variety of

carriers available, each with links back to one of the main branches of natural science: biology, chemistry,

mikos: “9026_c014” — 2007/4/9 — 15:51 — page3—#3

Gene Therapy 14-3

TABLE 14.1 Examples of Virus Families and Members Used for

Gene Delivery

Family Example References

Adenoviridae Adenovirus [14]

Baculoviridae Baculovirus [15]

Herpesviridae Pseudorabies virus [16]

Simplex virus [17]

Parvoviridae Adeno-associated [18]

Parvovirus [19]

Poxviridae Vaccinia [20]

Yaba monkey tumor virus [21]

Retroviridae Human immunodeﬁciency virus 1 and 2 [22,23]

Lentivirus [24]

Murine leukemia virus [25]

Retrovirus [26]

and physics. It is possible for hybrid systems that combine two or more basic technologies to exist, but the

individual classiﬁcations will be discussed separately for clarity.

14.3.1 Biological Delivery Methods

Because they are a product of millions of years of biologic evolution, viruses are included here as the

biological forms of gene delivery in spite of the fact that viruses are not technically alive (they do not

respire). Viruses are the most efﬁcient vectors for large-scale gene delivery. Some also offer permanent

expression of delivered genes through the integration of genetic material into the transduced organism’s

genome. Table 14.1 lists some of the major viral families and speciﬁc members that have been used in

gene therapy. Many of the references in the table cite review articles of the speciﬁc virus classiﬁcation for

further reading.

Currently, herpes simplex virus (HSV) is being used to transduce cells in the central nervous system.

By altering the genome of the virus, researchers have been able to decrease cytotoxicity and increase

transfection efﬁciency [27]. HSV is also desirable because it is relatively large to allow packaging of larger

plasmids, does not integrate its DNA into the host genome which avoids the deleterious effects that are

possible with random integration, and is easily deliverable into the brain due to its ability to actively travel

towards the nuclei of the neurons through the axons in the peripheral nervous system. HSV has also been

applied to the transduction of skeletal muscle. Recent reports have described successful HSV-mediated

gene transfer into skeletal muscle cells in vitro and in vivo and outlined possible applications for the

delivery of large genes such as that coding for dystrophin [28].

In the liver, the virus AcMNPV (of the family baculoviridae) has yielded promising transfection efﬁ-

ciencies [29]. The YABA-like virus, which exhibits very similar attributes to the vaccinia virus but does not

produce immune responses in the host, has successfully targeted and treated ovarian cancer in mice [30].

Adeno-associated virus (AAV) requires coinfection with a helper virus (typically adenovirus) to be pro-

ductive. AAV offers an advantage to the gene therapist in that it has been reported to provide permanent

expression of delivered genes. In utero experiments in mice and nonhuman primates indicate that recom-

binant AAVs can successfully alter the genome of the host organism to produce stable transductants [31].

Adeno-associated viruses are commonly used and successful in targeting the central nervous system [32].

Although repeated viral transduction is associated with inﬂammatory and immunological responses in

the host, it has been reported that these responses can be reduced by carefully balancing recombinant AAV

dosing with prudent timing [33].

Retroviruses offer good transduction efﬁciencies in vivo. Hallmarks of retroviral transduction are that

RNA is delivered into cells as opposed to DNA, cDNA is made using viral reverse transcriptase, and

this cDNA is introduced into the host genome via the protein integrase to produce stable transfect-

ants. Immunodeﬁciency viruses are commonly investigated retroviruses, and include HIV and feline

mikos: “9026_c014” — 2007/4/9 — 15:51 — page4—#4

14-4 Tissue Engineering

immunodeﬁciency virus (FIV). Investigations into retroviral genome alterations, such as self-inactivation

via promoter deletion, are being conducted in an attempt to render HIV-1 a safe vector for gene trans-

fer [34]. Use of FIV has shown efﬁcient transduction of nondividing cells without many of the inherent

risks associated with delivering recombinant HIV to humans [35]. Lentivirus, which belongs to the same

family as HIV and FIV, has been developed to produce concurrent regulated multiple gene delivery upon

transduction [36].

14.3.2 Chemical Delivery Methods

Many chemical methods of transfection have been engineered and utilized in the past 20 years. The

use of synthetic gene delivery vehicles, such as polymers or cationic lipids, offers several advantages.

Nonviral gene delivery is not restricted to plasmid DNA sizes based upon the ability to ﬁt into a viral

head of ﬁnite size, as is evidenced by successful delivery of a 2.3 Mb artiﬁcial human chromosome using

poly(ethylenimine)(PEI) [37], and a 60 Mb artiﬁcial chromosome into Chinese hamster ovary cells using

liposomes [38]. Although nonviral gene delivery usually results in transient gene expression, an advantage

of this characteristic is that there is little or no risk of random integration into host genomes. Random

integration poses a problem in that it can knock out a vital gene such as a housekeeping gene or a tumor

suppressor gene. An additional advantage of nonviral gene delivery is the reduced threat of immune

response in vivo versus repeated viral injections.

Several cationic polymers exist that exhibit strong transfection qualities. Many of these polymers are

based on polypeptide chains containing multiple lysine, histidine, or arginine residues. Historically,

poly(l-lysine) has been the most commonly used peptide transfection agent. However, several arginine-

based combinations of peptides have been formed into polyplexes of varying sizes and charge ratios which

may yield better transfection results than the previously used polypeptide chains composed of a single

amino acid [39]. Histidine–lysine polyplexes have also produced good transfection results that positively

correlated with the amount of histidine in the complex [40]. There are also additions that can be made

to these complexes in order to increase transfection efﬁciency and lower cytotoxic effects. These include

ligands such as nerve growth factor, and hydrophilic polymers such as polyethylene glycol (PEG), which

has been shown to increase transfection efﬁciency and lower cytotoxicity of poly(l-lysine) both in vitro

and in vivo [41].

PEI is a highly cationic polymer that is available in both linear and branched forms. Each form of the

polymer has a repeating [–CH

–CH

–N<] structure, with each nitrogen taking the form of a primary (end

group), secondary (middle of a straight chain), or tertiary (branch point) amine (Figure 14.1). The groups

attached to secondary and tertiary amines are typically additional [–CH

–CH

–N<], although terminal

amines can be modiﬁed through the attachment of moieties for targeting or degradation purposes. Because

of the large number of amines present in PEI, many of which carry a positive charge at physiological pH,

DNA and RNA easily bind with the polymer via electrostatic interactions to form stable complexes.

The transfection efﬁciency of branched PEI correlates with its molecular weight, with weight-average

molecular weights of approximately 25,000 Da working best [42,43]. PEGylation of PEI polymers can,

as was also the case for poly(l-lysine), increase transfection efﬁciency; this has been indicated in delivery

of complexes to the central nervous system in vivo [44]. PEI has also been successfully used in in vitro

transfections of rat endothelia and chicken embryonic neurons as well as in vivo in mice [45]. Conjugates

of PEI are also being developed, such as silica microspheres coated with the polymer–DNA complexes.

These conjugates have been used successfully for in vitro transfection, and offer the potential for relatively

simple covalent surface modiﬁcations [46].

Dendrimers are highly branched polymers built around a single atom or molecule. Poly(amidoamine)

(PAMAM) dendrimers are often used for gene delivery because of their high nitrogen content which

aids in DNA binding and condensation. With a central atom of nitrogen, these polymers are built by

the addition of amine-containing molecules, such as [CH

–CH

–NH

] (Figure 14.1). The result is a

maximally branched molecule of known molecular weight; a sort of controlled version of PEI. PAMAM

dendrimers, sometimes referred to as starburst dendrimers, have shown good transfection efﬁciencies in

mikos: “9026_c014” — 2007/4/9 — 15:51 — page5—#5

Gene Therapy 14-5

-CH

-NH

C-CH

-NH CH

-CH

-NH

(a)

(b) (c)

FIGURE 14.1 Three polymers based upon the [–CH2–CH2–N<] basic unit. Amines are shown in the uncharged

state for clarity. (a) Linear PEI; (b) An example of a branched PEI. The polymerization permits 2

◦

amines and one

cyclization via a back biting reaction. The polymer is somewhat irregular; (c) Third generation PAMAM dendrimer.

All amines are tertiary amines (except for chain termini).

mammalian cells with little or no cytotoxicity in vitro [47]. A current application of this dendrimer is its

coupling with an Epstein–Barr virus-based plasmid for suicide gene therapy in cancer cells [48].

Chitosan is another polymer that has been well characterized for use in transfection [49]. This nat-

ural nontoxic polysaccharide lends DNAase-resistance to its cargo while condensing the DNA to form

stronger complexes [50]. The efﬁciency of chitosan is thought to rely upon its ability to swell and burst

endolysosomes, which allows the delivered DNA to continue its path to the nucleus [51].

Alginate, a polycationic polysaccharide that can be used in the form of a hydrogel, has been used

alone for transfection [52], as well as in combination with poly(l-lysine) complexes to form an

oligonucleotide “sponge” [53]. Through slow degradation, these gels were shown to release embedded

oligonucleotides over time. Possible applications of these gels to deliver antisense RNA are currently being

pursued [53].

Microgels are similar to the alginate complex just mentioned. A thermosensitive microgel, composed of

poly[ethylene glycol-(d, l-lactic acid-co-glycol acid)-ethylene glycol] (PEG–PLGA–PEG) triblock copoly-

mers, has been created to have the property of being a liquid at room temperature but solidifying at

◦

C. Once solidiﬁed, the gel slowly degrades over the course of 30 days, releasing its component oligo-

nucleotides to the surrounding cells. Possible applications of this gel system to wound healing are being

pursued [54].

Lipids, particularly cationic lipids, have been used for gene delivery in a process termed lipofection. Sev-

eral lipofection agents have been used for successful transfection of cells both in vitro and in vivo. Because

of the aqueous environment inside cells and tissues, the hydrophobic tails of the lipids will coalesce to form

hollow micelles, the interiors of which can contain oligonucleotides for cellular delivery. The combination

of more than one hydrophobicentity can often yield higher transfection efﬁciencies than using a single type

of lipid. In primary cortical neurons, a combination of N -[1-(2,3-dioleoyloxy)propyl]-N,N ,N -trimethyl-

ammonium methylsulfate and cholesterol (DOTAP : Chol) shows high transfection efﬁciency [55]. Using

intraveneous administration of N-[1-(2,3-dioleoyloxy)propyl]-N ,N ,N -trimethylammonium chloride–

Tween 80 (DOTMA-Tween 80) complexes with high DOTMA : DNA ratios, transient transfection to

several organs has also been accomplished [56]. The use of helper lipids such as cholesterol or dioleoyl

phosphatidylethanolamine (DOPE) can increase transfection efﬁciency signiﬁcantly through endosomal

membrane destabilization [57].