An Introduction to Molecular Medicine and Gene Therapy - part 6 ppsx

In addition, angiogenic gene therapy may offer an adjunct to traditionaltherapies that improves long-term outcomes.GENE THERAPY OF VASCULAR GRAFTS Modification of Vein Graft Biology The

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factors or the genes encoding these factors have been administered to a smallnumber of patients These studies have involved either the use of angiogenic factorswith peripheral vascular or coronary artery disease in patients who were not can-didates for conventional revascularization therapies or the application of proan-giogenic factors as an adjunct to conventional revascularization The modest doses

of either protein factors or genetic material delivered in these studies were not ciated with any acute toxicities Concerns remain, however, regarding the safety ofpotential systemic exposure to molecules known to enhance the growth of possibleoccult neoplasms or that can enhance diabetic retinopathy and potentially evenocclusive arterial disease itself Despite early enthusiasm, there is little experiencewith the administration of live viral vectors to a large number of patients Thus, it

asso-is uncertain whether potential biological hazards of reversion to competent states or mutation and recombination will eventually become manifest

replication-In addition, it is also unclear whether the clinical success of conventional cularization, which has involved the resumption of lost bulk blood flow throughlarger conduits, will be reproduced via biological strategies that primarily increasemicroscopic collateral networks It must also be remembered that neovasculariza-tion is itself a naturally occurring process The addition of a single factor may notovercome conditions that have resulted in an inadequate endogenous neovascular-ization response in patients suffering from myocardial and lower limb ischemia.Despite these limitations, angiogenic gene therapy may provide an alternative notcurrently available to a significant number of patients suffering from untreatable

revas-GENE THERAPY FOR ANGIOrevas-GENESIS 191

FIGURE 8.3 Combined gene transfer and transmyocardial laser revascularization (TMR) See color insert Schematic representation of chronic ischemia induced by placement of Ameroid constrictor around the circumflex coronary artery in pigs Ischemic hearts that underwent TN4R followed by injection of plasmid encoding VEGF demonstrated better normalization of myocardial function than either therapy alone.

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disease In addition, angiogenic gene therapy may offer an adjunct to traditionaltherapies that improves long-term outcomes.

GENE THERAPY OF VASCULAR GRAFTS

Modification of Vein Graft Biology

The long-term success of surgical revascularization in the lower extremity and nary circulations has been limited by significant rates of autologous vein graft failure

coro-A pharmacologic approach has not been successful at preventing long-term graft eases such as neointimal hyperplasia or graft atherosclerosis Gene therapy offers anew avenue for the modification of vein graft biology that might lead to a reduction

dis-in cldis-inical morbidity from graft failures Intraoperative transfection of the vedis-in graftalso offers an opportunity to combine intact tissue DNA transfer techniques with theincreased safety of ex vivo transfection A number of studies have documented thefeasibility of ex vivo gene transfer into vein grafts using viral vectors

The vast majority of vein graft failures that have been linked to the neointimaldisease is part of graft remodeling after surgery Although neointimal hyperplasiacontributes to the reduction of wall stress in vein grafts after bypass, this processcan also lead to luminal narrowing of the graft conduit during the first years afterthe operation Furthermore, the abnormal neointimal layer, producing proinflam-matory proteins, is the basis for an accelerated form of atherosclerosis that causeslate graft failure

As in the arterial balloon injury model, a combination of antisense ODN ing expression of at least two cell cycle regulatory genes could significantly blockneointimal hyperplasia in vein grafts Additionally, E2F decoy ODN yield similarefficacy in the vein graft when compared to the arterial injury model In contrast toarterial balloon injury, however, vein grafts are not only subjected to a single injury

inhibit-at the time of operinhibit-ation, but they are also exposed to chronic hemodynamic stimulifor remodeling Despite these chronic stimuli, a single, intraoperative decoy ODNtreatment of vein grafts resulted in a resistance to neointimal hyperplasia that lastedfor at least 6 months in the rabbit model During that time period, the grafts treatedwith cell cylce blockage were able to adapt to arterial conditions via hypertrophy

of the medial layer Furthermore, these genetically engineered conduits provedresistant to diet-induced graft atherosclerosis (Fig 8.4) They were also associatedwith preserved endothelial function

An initial prospective, randomized double-blind clinical trials of human vein grafttreatment with E2F decoy ODN has recently been undertaken Efficient delivery

of the ODN is accomplished within 15 min during the operation by placement ofthe graft after harvest in a device that exposes the vessel to ODN in physiologicsolution This device creates a nondistending pressurized environment of 300 mmHg(Fig 8.5) Preliminary findings indicated ODN delivery to greater than 80% of graftcells and effective blockade of targeted gene expression This study will measure the effect of cell cycle gene blockade on primary graft failure rates and representsone of the first attempts to definitively determine the feasibility of clinical geneticmanipulation in the treatment of a common cardiovascular disorder

With the development of viral-mediated gene delivery methods, some

investiga-192 GENE THERAPY IN CARDIOVASCULAR DISEASE

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tors have begun to explore the possibility of using these systems ex vivo in gous vein grafts Studies have demonstrated the expression of the marker gene b-galactosidase along the luminal surface and in the adventitia of 3-day porcine veingrafts infected with a replication-deficient adenoviral vector for 2 h at the time ofsurgery Other studies have explored the use of a novel adenovirus-based trans-duction system in which adenoviral particles are linked to plasmid DNA viabiotin/streptavidin-transferrin/polylysine complexes b-Galactosidase expressionwas documented 3 and 7 days after surgery in rabbit vein grafts incubated for 1 hwith complexes prior to grafting Expression was greatest on the luminal surfaces

autolo-of the grafts The presence autolo-of transfected cells in the medial and adventitial layerswas also reported

The feasibility of gene transfer in vein grafts has subsequently lead to the

inves-GENE THERAPY OF VASCULAR GRAFTS 193

FIGURE 8.4 Control oligonucleotide-treated (A and B) and antisense oligonucleotide (against c and 2 kinase/PCNA)-treated vein grafts (C and D) in hypercholesterolernic rabbits,

6 weeks after surgery (¥7O) See color insert Sections were stained with hematoxylin/van Gieson (A and C) and a monoclonal antibody against rabbit macrophages (B and D) Arrows indicate the location of the internal elastic lamina.

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tigation of potential therapeutic endpoints such as neointima formation Studiesusing a replication-deficient adenovirus expressing tissue inhibitor of metallopro-teinase-2 (TIMP-2) demonstrate a decrease in neointimal formation in a saphenousvein organ culture model Other studies using intraoperative transfection of the

senescent cell-derived inhibitor (sdi, I) gene, a downstream mediator of the tumor suppresser gene p53 and the HVJ–liposome system, demonstrated a reduction in

neointima formation

Bioengineering and Gene Therapy

The use of gene transfer in vein grafts may go beyond the treatment of the graftitself The thrombogenicity of prosthetic materials, such as poly(tetrafluoroethylene)

194 GENE THERAPY IN CARDIOVASCULAR DISEASE

FIGURE 8.5 Intraoperative pressure-mediated transfection of fluorescent-labeled ODN to saphenous vein graft cells See color insert (A) Hoechst 33,342 nuclear chromatin staining

of vein graft in cross section, illustrating location of nuclei within the graft wall (100¥) (B) Same section of saphenous vein viewed under FITC-epifluoreseence at 100¥ Note the pattern of enhanced green fluorescence in the nuclei of cells within the graft wall, indicating nuclear localization of labeled ODN.

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(PTFE) or Dacron, has limited their use as small caliber arterial substitutes A bined bioengineering, cell-based gene therapy strategy may decrease this thrombo-genicity Successful isolation of autologous endothelial cells and their seeding ontoprosthetic grafts in animal models have been well characterized Furthermore, it hasbeen hypothesized that one can enhance the function of these endothelial cells viathe transfer of genes prior to seeding of the cells on the graft surface The initialreport of the use of this strategy achieved successful endothelialization of a pros-thetic vascular graft with autologous endothelial cells transduced with a recombi-

com-nant retrovirus encoding the lacz gene Successful clinical applications of these

concepts, however, have not been reported In an attempt to decrease graft bogenicity, 4-mm Dacron grafts were seeded with retroviral transduced endothelialcells encoding the gene for human tissue plasminogen activator (TPA) The graftswere subsequently implanted into the femoral and carotid circulation of sheep Theproteolytic action of TPA resulted in a decrease in seeded endothelial cell adher-ence, with no improvement in surface thrombogenicity

throm-GENE THERAPY FOR THE HEART

The myocardium has been shown to be receptive to the introduction of foreigngenes As seen in noncardiac muscle, measurable levels of gene activity has beenfound after direct injection of plasmids into myocardial tissue in vivo Althoughlimited to a few millimeters surrounding the injection site, these observations havelaid the basis for consideration of gene transfer as a therapeutic approach to cardiacdisease Additionally, both adenoviral and adenoassociated viral vectors can bedelivered to the myocardial and coronary vascular cells via either direct injection

or intracoronary infusion of concentrated preparations in rabbits and porcinemodels respectively Gene transfer into the myocardium has also been achieved viaeither the direct injection or intracoronary infusion of myoblast cells that have beengenetically engineered in cell culture

Congestive Heart Failure

The b-adrenergic receptor (b-AR) is known to be a critical player in mediating theionotropic state of the heart This receptor has received significant attention as atarget for genetic therapeutic intervention in congestive heart failure Transgenicmice were generated expressing the b2-AR under the control of the cardiac majorhistocompatibility complex (X-MHC) promoter These animals demonstrated anapproximately 200-fold increase in the level of b2-AR along with highly enhancedcontractility and increased heart rates in the absence of exogamous b-agonists Thisgenetic manipulation of the myocardium has generated considerable interest in theuse of gene transfer of the b-AR gene into the ailing myocardium as a means oftherapeutic intervention To date, attempts at exploring this exciting possibility havebeen primarily limited to cell culture systems However, recent studies have movethis technology into animal studies For example, adenoviral-mediated gene trans-fer of the human b2-AR successfully demonstrated improved contractility in rabbitventricular myocytes that were chronically paced to produce hemodynamic failure

An enhanced chronotropic effect resulting from the injection of a b2-AR plasmid

GENE THERAPY FOR THE HEART 195

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construct into the right atrium of mice has been performed But no evaluation ofenhanced contractility by transfer of this gene into the ventricle has been reported.These results demonstrate the feasibility of using the bP-adrenergic pathway andits regulators as a means by which to treat the endpoint effect of the variety ofcardiac insults.

There has also been recent interest in the enhancement of contractility throughthe manipulation of intracellular calcium levels Sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) transporting enzyme, which regulates Ca2+ sequestration into the sarcoplasmic reticulum (SR), has been shown to be decreased in a variety

of human and experimental cardiomyopathies Over expression of the SERCA2aprotein in neonatal rat cardiomyocytes using adenoviral-mediated gene transfer hasbeen achieved This leads to an increase in the peak (Ca2+li) release, a decrease inresting (Ca2+li) levels, and more importantly to enhanced contraction of the myocar-dial cells as detected by shortening measurements The success of this approach inimproving myocardial contractility has yet to be documented in vivo But onceagain, gene therapy approaches provide a novel and potentially exciting means bywhich to treat the failed heart

Myocardial Infarction

Myocardial infarction (MI) is the most common cause of heart failure At the lular level MI results in the formation of scar that is composed of cardiac fibrob-lasts Given the terminal differentiation of cardiomyocytes, loss of cell mass due toinfarction does not result in the regeneration of myocytes to repopulate the wound.Researchers have, therefore, pursued the possibility of genetically convertingcardiac fibroblasts into functional cardiomyocytes The feasibility of this notiongained support from gene transfer studies These studies used retroviral-mediatedgene transfer for the in vitro conversion of cardiac fibroblasts into cells resemblingskeletal myocytes via the forced expression of a skeletal muscle lineage-

cel-determining gene, MyoD Fibroblasts expressing the MyoD gene were observed to

develop multinucleated myotubes similar to striated muscle that expressed MHCand myocyte-specific enhancer factor 2 Additional studies have shown that the tran-

fection of rat hearts injured by freeze–thaw with adenovirus containing the MyoD

gene resulted in the expression of myogenin and embryonic skeletal MHC At thistime, however, functional cardiomyocytes have not yet been identified in regions ofmyocardial scarring treated with in vivo gene transfer

Ischemia and Reperfusion

Coronary artery atherosclerosis, and resulting myocardial ischemia, is a leadingcause of death in developed countries Reperfusion injury has been linked to significant cellular damage and progression of the ischemic insult In addition tostimulating therapeutic neovascularization, genetic manipulation may be used as ameans to limit the degree of injury sustained by the myocardium after ischemia andreperfusion The process of tissue damage resulting from ischemia and reperfusionhas been well characterized

Briefly, the period of ischemia leads to an accumulation of adenosine phate that then leads to increased levels of hypoxanthine within and around cells

monophos-196 GENE THERAPY IN CARDIOVASCULAR DISEASE

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in the affected area Additionally, increased conversion of xanthine dehydrogenaseinto xanthine oxidase takes place Upon exposure to oxygen during the period ofreperfusion, hypoxanthine is converted to xanthine This conversion results in thecytotoxic oxygen radical, superoxide anion (O2 -) This free radical goes on to formhydrogen peroxide (H2O2), another oxygen radical species Ferrous iron (Fe2+) accu-mulates during ischemia and reacts with H2O2, forming the potent oxygen radical,hydroxyl anion (OH-) These free radical species result in cellular injury via lipidperoxidation of the plasma membrane, oxidation of sulfhydryl groups of intracellu-lar and membrane proteins, nucleic acid injury, and breakdown of components ofthe extracellular matrix such as collagen and hyaluronic acid Natural oxygen radicalscavengers, such as superoxide dismutase (SOD), catalase, glutathione peroxidase,and hemoxygenase (HO) function through various mechanisms to remove oxygenradicals produced in normal and injured tissues.

The level of oxygen radical formation after ischemia–reperfusion injury in theheart can overwhelm the natural scavenger systems Thus, overexpression of eitherextracellular SOD (ecSOD) or manganese SOD (MnSOD) in transgenic mice hasimproved postischemic cardiac function and decreased cardiomyocyte mitochondr-ial injury in adriamycin-treated mice, respectively These findings suggest a role forgene transfer of natural scavengers as a means to protect the myocardium in theevent of an ischemia–reperfusion event Substantial protection has been observedagainst myocardial stunning, using intra-arterial injection of an adenovirus con-taining the gene for Cu/Zn SOD (the cytoplasmic isoform) into rabbits However,

no studies have investigated the direct antioxidant effect and ensuing improvement

in myocardial function of this treatment after ischernia and reperfusion injury Thisapplication of gene therapy technology may offer a novel and exciting approach forprophylaxis against myocardial ischemic injury when incorporated into a system oflong-term, regulated transgene expression

In addition to the overexpression of antioxidant genes, some researchers haveproposed intervening in the program of gene expression within the myocardiumthat lead to the downstream deleterious effects of ischemia reperfusion Forexample, the transfection of rat myocardium with decoy oligonulceotides, blockingthe activity of the oxidation-sensitive transcription factor NFk-B, may be a usefulapproach NFk-B is linked to the expression of a number of proinflammatory genes

It inhibition succeeded in reducing infarct size after coronary artery ligation.Genetic manipulation of donor tissues offers the opportunity to design organ-specific immunosuppression during cardiac transplantation Although transgenicanimals are being explored as potential sources for immunologically protectedxenografts, the delivery of genes for immunosuppressive proteins, or the blockade

of certain genes in human donor grafts, may allow site-specific, localized suppression Alternatively, these approaches could result in a reduction or elimina-tion of the need for toxic systemic immunosuppressive regimens Gene activity hasbeen documented in transplanted mouse hearts for at least 2 weeks after intraop-erative injection of the tissue with either plasmid DNA or retroviral or adenoviralvectors The transfer of a gene for either TGF-b or interleukin-10 in a small area ofthe heart via direct injection, succeeded in promoting immunosuppression of graftreject Cell-mediated immunity was inhibited and acute rejection was delayed Inanother study, the systemic administration of antisense ODN directed against inter-cellular adhesion molecules (ICAM-1) also prolonged graft survival and induced

immuno-GENE THERAPY FOR THE HEART 197

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long-term graft tolerance when combined with a monoclonal antibody against theligand for ICAM-1, the leukocyte function antigen.

SUMMARY

The field of gene therapy is evolving from the realm of laboratory science into aclinically relevant therapeutic option The current state of this technology has pro-vided us with an exciting glimpse of its therapeutic potential Routine application,however, will require improvement of existing techniques along with the develop-ment of novel methods for gene transfer More importantly, no one method of genetransfer will serve as the defining approach Rather, it will be the use of all avail-able techniques, either individually or in combination, that will shape the applica-tion of this therapy Over the past two decades, as scientists have begun to unlockthe genetic code, more insight into the pathogenesis of disease has been gained.With the use of gene manipulation technology, this new information can be used

to further improve the understanding and treatment of complex acquired and genital diseases previously unresponsive to traditional surgical and pharmacologictherapy

con-KEY CONCEPTS

• The ideal cardiovascular DNA delivery vector would be capable of safe andhighly efficient delivery to all cell types, both proliferating and quiescent, withthe opportunity to select either short-term or indefinite gene expression Thisideal vector would also have the flexibility to accommodate genes of all sizes,incorporate control of the temporal pattern and degree of gene expression, and

to recognize specific cell types for tailored delivery or expression

• Recombinant, replication-deficient retroviral vectors have been used sively for gene transfer in cultured cardiovascular cells in vitro, where cell pro-liferation can be manipulated easily Recombinant adenoviruses have becomethe most widely used viral vectors for experimental in vivo cardiovascular gene transfer Adenoassociated virus has successfully transduced myocardialcells after direct injection of viral suspensions into heart tissue; and these infections have yielded relatively stable expression for greater than 60 days.For nonviral gene delivery, the controlled application of a pressurized en-vironment to vascular tissue in a nondistended manner has recently been found to enhance oligonucleotide uptake and nuclear localization This method may be particularly useful for ex vivo applications, such as vein graft-ing or transplantation, and may represent a means of enhancing plasmid gene delivery

exten-• Gene therapy approaches using either cytostatic, in which cells are preventedfrom progressing through the cell cycle to mitosis, or cytotoxic, in which celldeath is induced, may inhibit neointimal hyperplasia of restenosis

• Gene therapy for therapeutic neovascularization targets angiogenic growthfactors

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• Gene therapy offers a new avenue for the modification of vein graft biology thatmight lead to a reduction in clinical morbidity from graft failures Intraoperativetransfection of the vein graft offers an opportunity to combine intact tissueDNA transfer techniques with the increased safety of ex vivo transfection.

• For gene therapy of the heart, genetic manipulation of the myocardium has generated considerable interest in the use of gene transfer of the b-adrenergicrecepter gene into the ailing myocardium as a means of therapeutic interven-tion For myocardial infarction, gene therapy offers the ability to geneticallyconvert cardiac fibroblasts into functional cardiomyocytes Genetic manipula-tion may be used to limit the degree of injury sustained by the myocardiumafter ischemia and reperfusion through the transfer of natural scavengers ofoxidative tissue injury

SUGGESTED READINGS

Cardiovascular Gene Therapy

Allen, MD Myocardial protection: Is there a role for gene therapy Ann Thorac Surg 68:1924–1928, 1999.

Amant C, Berthou L, Walsh K Angiogenesis and gene therapy in man: Dream or reality Drugs 59(Spec No 33–36), 1999.

Ponder KP Systemic gene therapy for cardiovascular disease Trends Cardiovasc Med 9:158–162, 1999.

Zoldhelyi P, Eichstaedt H, Jax T, McNatt JM, Chen ZQ, Shelat HS, Rose H, Willerson JT The emerging clinical potential of cardiovascular gene therapy Semin Interv Cardiol 4:151–65, 1999.

Vascular/Smooth Muscle Gene Therapy

Chang MW, Barr E, Lu MM Adenovirus-mediated over-expression of the cyclin/cyclin dependent kinase inhibitor, p2l inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty J Clin Invest 96:2260–2268, 1995.

Chang MW, Barr E, Seltzer J Cytostatic gene therapy for vascular proliferative disorders with

a constitutively active form of the retinoblastoma gene product Science 267:518–522, 1995 Dunn PF, Newman KD, Jones M Seeding of vascular grafts with genetically modified endothelial cells Secretion of recombinant TPA results in decreased seeded cell retention

in vitro and in vivo Circulation 93:1439–1446, 1996.

George SJ, Baker AH, Angelini GD Gene transfer of tissue inhibitor of

metalloproteinase-2 inhibits metalloproteinase activity and neointima formation in human saphenous veins Gene Therapy 5:1552–1560, 1998.

Gibbons GH, Dzau VJ The emerging concept of vascular remodeling N Engl J Med 330:1431–1438, 1994.

Houston P, White BP, Campbell CJ, Braddock M Delivery and expression of fluid shear stress-inducible promoters to the vessel wall:Applications for cardiovascular gene therapy Hum Gene Therapy 10:3031–3044, 1999.

Mann MJ, Gibbons GH, Tsao PS Cell cycle inhibition preserves endothelial function in genetically engineered rabbit vein grafts J Clin Invest 99:1295–1301, 1997.

SUGGESTED READINGS 201

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in Chapter 3), we deal with very delicate, complex networks of cells and face the issue of accessibility (Fig 9.1) and targeting the desired cell type(s) when considering gene therapy strategies in the central nervous system Unlike otherorgans in the body such as the liver or lungs where large proportions of the organs can be damaged with minimal or no functional consequences, damage toextremely small areas of the brain can be devastating Therapeutic targeting to selective areas or cell types will be difficult to achieve in the central nervous system (CNS).

Excluding the identified genetic causes of neurodegenerative diseases, the etiology underlying the primary neurological disorders is unknown While the prin-ciple cell types affected in disorders such as Parkinson’s and Alzheimer’s have been identified, the exact contributing factors or conditions that trigger relentlessneuronal degeneration are presently unknown Therefore, at this time, gene products that help to reduce the effects of neural dysfunction, offset neuronal death, inhibit apoptosis, or encourage cell survival form the basis of gene therapy

in the nervous system As gene therapy approaches are developed and refined,the outcome of gene therapy in the nervous system could be extremely effective

In this chapter, the key aspects of neural dysfunction associated with the nent nervous system disorders are explained Promising advances with gene trans-fer to the CNS have been made with different families of virus vectors A focus onthe vectors and the cells used for gene delivery in animal models is provided Impor-tant features of the clinical trials using genetically modified cells and trophic fac-

promi-203

An Introduction to Molecular Medicine and Gene Therapy Edited by Thomas F Kresina, PhD

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Cerebral cortex

Frontal lobe

Temporal lobe Cerebellum

Cerebral cortex

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tors for neurodegeneration are described, and we will illustrate how neuroscienceresearch in combination with genetics and molecular biology is guiding the future

of gene therapy applications in the nervous system

SORTING OUT THE COMPLEXITY OF THE NERVOUS SYSTEM

The nervous system is divided into two main parts: (1) the central nervous system consisting of the brain and spinal cord and (2) the peripheral nervous system(PNS) composed of the nervous tissue in the form of nerves that emerge bilaterallyfrom the brain and spinal cord that serve to keep the other tissues of the body

in communication with the CNS (Fig 9.2) Numerous types of neurons specialized

to receive, process, and transmit information via electrical impulses are primarilyresponsible for the functional characteristics of the nervous system (Fig 9.3).Neurons can be identified by their size, shape, development, and organization within the brain Neurons work in networks and secrete neurotransmitters and other chemical messengers at sites of functional contact called synapses At eachsynapse a region of the cell membrane in the presynaptic neuron is specialized forrapid secretion of one or more types of neurotransmitters This area is closelyapposed to a specialized region on the postsynaptic cell that contains the receptorsfor the neurotransmitter or other ligands The binding of the neurotransmitter tothe receptors triggers an electrical signal, the synaptic potential, in the postsynapticcell (Fig 9.4) Information in the nervous system is thereby transmitted and pro-cessed by elaborate networks that generate a spectrum of electrical and chemicalsignals

Glial cells, often referred to as specialized support cells of the CNS, represent thesecond major class of cells that perform important functions that are key to thenormal operation of the nervous system (Fig 9.3) There are four main types of glialcells Astrocytes act in a general supportive capacity and help to maintain the extra-cellular environment in the CNS The astrocyte processes are intimately associatedwith the neuronal cell bodies, dendrites, and nerve terminals They serve to insulateand isolate pathways and neuronal tracts from one another Oligodendrocytes andSchwann cells form the myelin sheaths around axons in the CNS and PNS, respec-tively The myelin is wrapped around segments of axons and serves to accelerate theconduction of the electrical signals In the CNS, each oligodendrocyte may form andmaintain myelin sheaths for approximately 60 axons In the PNS, there is only oneSchwann cell for each segment of one axon Microglial cells in the CNS are analo-gous to macrophages and can be activated by a number of conditions, includinginflammation and trauma

SORTING OUT THE COMPLEXITY OF THE NERVOUS SYSTEM 205

FIGURE 9.1 External view of the cerebral hemisphere (a) Brain and spinal cord are

pro-tected by many layers including the skin, bone, and special connective tissue layers referred

to as the meninges (b) Schematic diagram of the protective layers that cover the brain (c) Major divisions of the human brain as seen from a midsaggital view.

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206 COMPONENTS OF CELL AND GENE THERAPY FOR NEUROLOGICAL DISORDERS

Posterior view The peripheral nerves in humans

C1 C2 C4 C5 C7 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 L1 L2 L3 L4

S1 S3 S5 C1

Thoracic nerves Spinal cord Cervical nerves

Sacrum

FIGURE 9.2 Brain, spinal cord, and peripheral nerves There are 31 vertebral bones in the spinal column that house and protect the spinal cord Between the vertebrae, spinal (peripheral) nerves emerge bilaterally The individual nerves are made of sensory and motor fibers that interface the peripheral parts of the body with the central nervous system (brain and spinal cord).

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WHAT GOES WRONG IN NEUROLOGICAL DISORDERS?

Given the vast number and types of neurons and glial cells in the nervous system,one quickly realizes the potential for several neurological dysfunctions, depending

on the cell type(s) affected Neuronal degeneration can occur in selected areas ofthe brain or neurodegenerative events may affect the entire brain (global neu-

WHAT GOES WRONG IN NEUROLOGICAL DISORDERS? 207

Dendrites

Axon

Oligodendrocyte

(glia) Myelin sheath

Synapse

Motor neuron Axon

Myelin

Oligodendrocyte cell cytoplasm

Direction of action potential

Neuron cell body

Astrocytes (glia)

FIGURE 9.3 Schematic representation of neurons and glial cells Neurons are surrounded

by astrocytes that fill the interstices between neuronal cell bodies Glia outnumber neurons

by at least 10 to 1 Oligodendrocytes wrap around the axon and produce the myelin sheath Inset shows how the myelin wraps around segments of the axon.

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rodegenerative conditions) as in the case of the neurogenetic lysosomal storage diseases (LSD) associated with single-gene mutations.

For the majority of neurological disorders, specific classes of neurons in the brain

or spinal cord show selective vulnerability Depending on the type of neuron/neurotransmitter affected, changes will occur in behavior, memory, or movement

In Parkinson’s, neurons located in the substantia nigra of the midbrain that contain

208 COMPONENTS OF CELL AND GENE THERAPY FOR NEUROLOGICAL DISORDERS

Axon Microtubules

Synaptic vesicle

Synaptic cleft Dendrite

Channel

K+

Action potential

Channel opens.

Reuptake of neurotransmitter by presynaptic neuron or astrocytes

The flow of sodium ions (Na+) and potassium ions (K+) generates a new electrical signal

FIGURE 9.4 Components of a synapse Illustration shows aspects of neurotransmitter release, receptor interaction, and generation of the electrical signal All electrical signals arise from the action of various combinations of ion channel proteins that form aqueous pores through which ions traverse the membranes When ion channels are open, ions move through the channels down their electrochemical gradients Their net movement across the membrane constitutes a current that changes the membrane potential and generates an electrical signal.

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the neurotransmitter dopamine undergo accelerated cell death Loss of theseneurons influences the normal function of the extrapyramidal system in the brainand results in rigidity and tremor of the limbs Alzheimer’s isolates the hippocam-pus and regions of the cerebral cortex due to death of acetylcholine-rich neurons,causes dementia, and prevents the formation of new memory Amyotrophic lateral sclerosis (ALS) damages the motor neurons in the CNS and causes weakness andspasticity Alternatively, when oligodendrocytes in the central nervous system areaffected, problems develop with routine motor functions, and sensory deficitsbecome noticeable in individuals with multiple sclerosis.

The LSD are genetic disorders resulting from mutations in genes that code forproteins involved with the degradation of normal body compounds that includelipids, proteins, and carbohydrates Although most lysosomal disorders result fromdefects in genes that code for lysosomal enzymes, some are caused by genes codingfor transport proteins, protective proteins, or enzymes that process the lysosomalenzymes Individually, the LSD occur infrequently, but collectively they occurapproximately in 1/5000 births The accumulation of enzyme substrates in cells

of the CNS characterizes disorders like the mucopolysaccharidoses or GM1

We have identified various types of cytological and molecular changes in neuronsthat are associated with the death of neurons Research has identified numerous,specific changes in neurons at risk associated with the prevalent CNS disorders andalso with the aging process Abnormal accumulations of filaments and altered pro-teins are recognized as primary features of neurons targeted in neurological dys-function The accumulations may occur in the cytoplasm of the neuron or in theextracellular environment In certain instances, the pattern of neuronal loss is dic-tated by how the neurons are connected to one another Alzheimer’s is an excellentexample of this point Virtually all the subgroups of neurons lost in Alzheimer’s are found to be connected to regions of the cerebral cortex that show high levels

of neuritic plaque formation—foci of degenerating processes and twisted arrays ofcytoskeletal elements in the neurons referred to as neurofibrillary tangles

What sets off the initial changes in neurons that lead to a cascade of cell death

in specific areas and pathways of the nervous system? A number of molecular anisms at different levels of neuronal function have been proposed Changes to thecytoskeleton, oxidative injury, deoxyribonucleic acid (DNA) modifications, changes

mech-in ribonucleic acid (RNA)/protemech-in synthesis, abnormal protemech-in accumulation, free radicals, reduced axonal transport, and programmed cell death have been iden-tified as possible reasons for neurological disease Several animal models are used

toxic-to generate these molecular changes, and, in turn, they help define the possible etiology of neurodegeneration and provide a way to test gene therapy strategies for CNS disorders, injury, or aging

WHAT GOES WRONG IN NEUROLOGICAL DISORDERS? 209

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NEUROTROPHIC FACTORS AND GENE THERAPY

Neurotrophic Factors

There are a variety of molecules in the nervous system that are important to thesurvival, differentiation, and maintenance of neurons in both the PNS and CNS.These molecules, referred to as neurotrophic factors (Table 9.1), induce pattern andsynapse formation and create highly specialized neural circuits in the brain Thefactors are secreted from the target innervated by the neurons, taken up at the nerveterminals, and then transported over long distances to the cell body where they act

to regulate neuronal functioning by a variety of signaling mechanisms (Fig 9.5) Wenow realize that neurotrophic factors bind to cell surface receptor proteins on the nerve terminals, become internalized (receptor-mediated endocytosis), and then move toward the cell body by the mechanism of retrograde axonal transport.Advances in the understanding of the structure of the receptors for neurotrophicfactors indicate that they are similar to the receptors used by traditional growthfactors and cytokines The expression of the receptors for the neurotrophic factors

is exclusively or predominantly in the nervous system, and, when activated, thefactors display distinctive molecular actions

Nerve growth factor (NGF) is the prototype member of the neurotrophins, afamily of proteins that have common structural features It was discovered and char-acterized in the 1950s by Rita Levi-Montalcini, Stanley Cohen, and Viktor Ham-burger and was the first molecule to show potent nerve growth promoting activity

on explants of neural tissue maintained in tissue culture Since the discovery of NGF,

a number of molecules have been identified and added to the expanding list of substances grouped under the broad umbrella of neurotrophic factors Common,well-studied factors are listed in Table 9.1 Responses to the neurotrophins are medi-

ated through receptor tyrosine kinases that belong to the trk family of

protoonco-210 COMPONENTS OF CELL AND GENE THERAPY FOR NEUROLOGICAL DISORDERS

TABLE 9.1 A Listing of Common Neurotrophic Factors

LIFRb/TYK

IGF-2

aFGF

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genes It is now clear that neurotrophic factors can be provided by a number ofsources including glial cells, afferent processes of neurons, muscle, and even by theextracellular matrix Numerous biological events including neuronal growth, phe-notype (neurotransmitter) expression, and programmed cell death have been linkedwith retrograde neurotrophic factor signaling Hence, there are many possible lines

of study to explore the effects of neurotrophic factor gene therapy in relation tobasic neural cell survival and function for the treatment of neurodegenerative disorders

From basic research, we have learned that if the brain is injured, these moleculescan be released to play a significant role in the recovery process In addition to limiting the loss of neurons, neurotrophic factors can stimulate new outgrowth fromthe axons and dendrites, regulate axon branching, modulate neurotransmitter synthesis, and influence synapse formation This inherit property of structural and functional change in neurons in response to environmental cues (like the release

of neurotrophic factors) is referred to as plasticity Many factors have been shown

to have overlapping effects (primarily on development and survival) on subsets

of neurons in the central and peripheral nervous system It is now very clear thatany given type of central or peripheral neuron needs a combination of factors, ratherthan a single neurotrophic factor to optimize survival and function Therefore,decisions must be made regarding the most effective combinations of factors for the neurons/neurological disorder in question As discussed later in this chapter,

NEUROTROPHIC FACTORS AND GENE THERAPY 211

FIGURE 9.5 Retrograde signaling by neurotrophic factors The neurotrophic factor ligand (supplied by a target tissue) binds to the receptor on the surface of the axon terminal This receptor–ligand complex is then transported along the axon to the cell body Retrograde trophic signals have been shown to modulate neuronal growth, survival, death, and the expression of neurotransmitters.

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