2 Neurotransplantation and Gene Transfer

2.1 Introduction

Research on the nervous system has been intensified over the last decades and still is booming. Basic neuroscience has dramatically increased the knowledge of cellular and molecular mechanisms of brain functioning. As a result cellular and molecular disarrangements that go with the variety of neurological, neurodegenerative and psychiatric disorders have been identi- fied, although the primary causes of these disorders remain largely unknown. In cases of acquired or degenerative loss of nervous functions, pharmacological treatments are still mainly directed towards the ameliora- tion of symptoms and the limitation of secondary tissue damage. This clini- cal problem together with the development of new potential therapeutic techniques such as cell grafting or implantation and gene transfer, have led to the exploration of interventions in the nervous system that can tentatively be called “restorative neurosurgery”. A shift from animal experimentation to clinical trials occurred rapidly and experimental neurotransplantation sur- gery in patients with neurological and neurodegenerative disorders has taken place over several decades. Neuroscience, however, is just beginning to explore cellular and molecular interventions in diseased, degenerating or traumatised human nervous systems. Are we approaching an era of inter- ventions in the brain that will revolutionise treatment of thus far untreatable brain disorders? And if so, how do we ensure that these interventions will be performed in a safe, adequate and acceptable way? How do we judge and prevent the risks of unwanted changes in brain function and in the human psyche?

2.1.1 Restorative Neurosurgery by Cells and Genes

In particular, neural tissue grafting has attracted great interest for its poten-

tial as a treatment for human neurodegenerative diseases. The basis for this

was the discovery that immature nerve cells (neurons) can survive implanta-

tion in the brain of laboratory animals, something which adult neurons are

unable to do. Immature nervous tissue taken from unborn foetuses can

develop normal neuronal properties in the adult nervous system. Immature

neurons grow and differentiate and form functional contacts with host brain

neurons. The grafting of human embryonic nerve cells would therefore

allow replacement of lost neurons in the case of a disease like Parkinson’s

disease in which the loss of so-called nigro-striatal dopaminergic nerve cells

seems to be a primary cause. They might also allow reconstruction of a brain

circuit like that of the striato-frontal system, effected by Huntington’s dis-

ease, through the supplementation of the atrophic striatal spiny neurons.

However, the neuronal loss in such neurodegenerative diseases may also be ameliorated or even stopped by the implantation of cells that provide trophic support in order to keep the neurons at risk alive. These cell implants should do so by the release of neuro protective or neuralgrowth-stimulatory proteineous compounds. Such factors can not be applied systemically due to a short half life in vivo and the need to apply them locally to prevent any negative side effects on nearby intact systems. The genetic modification of the affected cells, or their neighbouring cells to let them locally produce such factors has also been proposed as a possibility. This does not involve a cell implantation but treatment involving a “gene transfer by injection”. Cell implantation and genetic modification may even be combined when the implanted cells are modified to express a therapeutic gene. Cell and gene therapy for the diseased or traumatised brain, however, are both invasive interventions in the human brain, the organic basis of our personhood. Such interventions cannot be reversed or halted in the way that a patient is able to stop taking a drug. Even if the treatment has a built-in termination process that can kill the implanted or genetically modified cells in case of unwanted side effects arising, or when the prescribed period of therapy should end, the organisation of the brain at the point of termination is never the same as that prior to intervention.

2.1.2 The Brain is Seat of the Human Mind

There is no doubt that the central nervous system (CNS), and in particular the brain, is the “seat” of the biological processes that underlie human iden- tity and personality, and thus a person’s character and mental capacities. It is of central importance for our behaviour, perceptions, thoughts and feelings and regulates body functions such as heart rate, muscle responses and con- trol of our immune system. It works in conjunction with both our bodies and the outside world. It is the organ of our personhood and, in this respect, it is a unique and indispensable organ for human self-consciousness.

Alter- natively, one can say that our mind is our brain in action. Without this action the human mind is gone.

Small differences in the structure and organisation of the brain lead to different functional capacities and, therefore, to differences in mental and physiological processes directed by and derived from the brain. Such differ- ences underlie differences in personality between individuals. These differ- ences are only partially determined by the genotype as even identical twins differ in functional capacities and personality. Neurodegenerative and trau- matic disorders obviously alter the organisation of the nervous system and,

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This uniqueness is further explained by the (albeit fictional) thought experiment

of human brain transplantation. The result will be that the brain donor will see

him-/herself as having received a new body. On the other hand, the recipient ‘per-

son’ will cease to exist. Brain transplantation, therefore, does not exist or should

be defined as body transplantation.

therefore, the functioning of the brain. Cell implantations in the brain and gene transfer to brain cells which aim to restore brain functions will never be able to restore the structure and organisation of the brain prior to the impact of the disease or trauma. Therefore, unwanted physiological and mental side effects will occur on the recipient of any form of intervention. However, these side effects may be very subtle and will not necessarily be obvious in normal daily life.

This chapter will focus on interventions in the brain in the field of restorative neurosurgery. The goal of these interventions is tissue repair or the introduction of physical changes in brain chemistry in order to relieve the symptoms of certain diseases. Neurodegenerative diseases like Parkin- son’s disease (PD), Huntington’s disease (HD), Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS), as well as acquired nervous trauma as the result of a stroke or spinal cord injury, are the first candidate diseases for such approaches. The neural grafting of fetal brain cells (neurotransplantation) in PD and HD patients was the first experimental clinical treatment explored because precise aims could be for- mulated for these interventions (implanting nerve cells to deliver dopamine and supplementing interneurons in the striatum of the brain in the respec- tive cases). Clinical research is less advanced for other neurodegenerative diseases and in the field of brain trauma, where the target for restoration is larger or less well-known. In these cases, the potential target for intervention in the CNS is largely unknown and this is crucial knowledge for any restora- tive neurosurgery. In principle, if a brain disorder can be pinpointed to a particular (local) cellular or molecular origin, or a target site for regenera- tion can be identified, then cell or gene therapy may be possible. For psychi- atric disorders incompatible with normal life, which are potentially caused by a multiplicity of interrelated factors, cellular and molecular interventions in the brain may not be feasible unless one finds a specific target which can be reached through the efficient treatment of a particular sub-symptom.

This may not be unrealistic as nowadays the deletion of single genes in trans- genic animals can show dramatic behavioural effects in areas such as drug dependency, anxiety, depression and fear conditioning, indicating the possi- bility of existing primary targets.

2.1.3 Questions to be Answered

Several interconnected ethical and normative questions arise from clinical

neurotransplantation and gene transfer. When and how should a brain dis-

order be subject to (safe) cellular or molecular intervention? How can we

design meaningful and morally acceptable experimental studies of the

effects of these procedures on human beings? What criteria should be

employed to evaluate the outcome of experimental restorative neuro-

surgery? What should be the correct procedures for the retrieval and use of

embryonic tissue from aborted fetuses or collected from organ donor?

What criteria should govern the use of human stem cells when derived from in vitro human blastocysts (their stem cells are nowadays seen as a very potent source of cells for tissue repair of all organs thereby providing a future solution for many life-threatening organ failures, including brain failures)? Should we permit the use of cells from prenatal animal sources in order to circumvent the ethical problems surrounding the use of human fetuses? Can genetic modification be safely established? Finally, given the view that the CNS will be altered by an intervention, how do we weigh up the benefits and the risks of any such procedure for the patient? Before dis- cussing these points, it seems appropriate to shortly outline the organisa- tion of the brain and its principle functional processes as well as the current status of experimental neurotransplantation and gene transfer in human patients.

2.2 Brain Structure and Capacities

The nervous system consists of a central part and a peripheral part. The nerves arising from the spinal cord and the nodes of certain neurons in the body (ganglia, retina, and olfactory epithelium), serve to innervate the organs, muscles and skin and comprise the peripheral nervous system (PNS). The brain and the spinal cord are referred to as the central nervous system (CNS), whereby the spinal cord is the means through which the CNS is able to communicate with the PNS. The CNS receives input from the body and from the environment through sensory, auditory and visual organs as well as chemicals (including hormones). This information is processed and either generates an output in the form of bodily, emotional, cognitive and anticipatory reactions or is kept and stored as memory for future challenges.

The individual brain acquires and accommodates these mechanisms in its developmental period, but these mechanisms remain subject to active self- organising and reorganising changes throughout life.

2.2.1 Neurons Act in Networks

The basic units of the nervous system are the neurons. These cells have a

cell body and many cell processes (neurites), one of which is the axon that

transfers a pulse to other neurons or to non-neuronal targets outside the

nervous system. The message to other neurons is carried in chemical com-

pounds – neurotransmitters – that are released at the contact zone of the

axon, the synapse. Examples of neurons having a peripheral target rather

than other neurons are motoneurons (innervate the muscles) and neu-

rosecretory neurons (releasing hormones into the circulation). The release

of the emitted messenger is always evoked by so-called action potentials, a

moving change in electric membrane potential arising at the cell body and

traveling along the axon. The sum of these action potentials of individual

neurons as well as the continuous changes in the membrane potential of

the neurons, make up the electro-encephalogram (EEG) which can be measured by placing electrodes on the surface of the skull. The human brain contains about 10

neurons (about 20 times the world population of humans). Each neuron receives the input of an average of 1,000 synapses on their neurites (having an estimated total length of 100,000 km in the human brain). This is an amazing number of nodes per cell and within the CNS for internal brain communication. Neurons by themselves are functionless units. However grouped, either in nuclei or in layer struc- tures, neurons make up networks that mutually interact to form informa- tion-processing units. Within a substructure of the brain, different types of neurons have different connections and use different chemical com- pounds for signal transfer. These neurotransmitters reach different types and quantities of receptors on the target site of the synapse, thereby affect- ing intracellular molecular machinery in the receiving cells. For a single neuron, the mix of incoming signals will be integrated, and will lead either to the adaptation of cell functions or to an action potential via its axon, i.e., a message to its target (after a change of its membrane potential above a certain threshold). In addition, the human brain contains glial cells.

There are around ten times as many of these than the total number of neu- rons in the brain. These cells are involved in the regulation of signal trans- duction. Various subtypes of glial cells have different functions among which the electric isolation of axons is but one. The result of all the inter- actions of neurons in the human brain steers or directs both body func- tions and the psyche.

2.2.2 Networks are Formed by Genotype and Environment Different groups of neurons are incorporated into different networks of functional activities in the brain known as neuronal systems. If parts of these systems or their connections are lost (as in the case of degeneration or trauma) or are not properly working (as the result of toxins or the transient overexposure to neuro-active compounds), the functional capacity of the system is affected. The neuronal cell acquisition of the human nervous sys- tem takes place both before and after birth, and continues in young children up to approximately three years of age. Neuronal networks are formed throughout this period too but continue to be formed up to approximately 18 years of age, i.e. until after puberty, when the nervous system is said to be fully matured. Brain development is an orchestrated process whereby the

“birth” of neurons during fetal life is genetically defined, but the differentia- tion, maturation and organisation of neurons depends on the appearance of other groups of neurons and on molecular signals from non-neuronal cells.

Thus, the connectivity of neurons depends on the temporal profile of partic-

ular signals. During development, neurons are often born in excess and

compete for survival by establishing connections with their targets (see fig-

ure 2.1).

The early period of development of the brain is very sensitive to the exter- nal factors that may interfere with brain cell activity. It is claimed that any circumstance known to interfere with the functioning of the adult nervous system, when imposed on the developing brain, will (or at least could) alter the functional capacities of the brain permanently (Swaab and Boer 2001).

In other words, the regulatory systems of neuronal circuits in the mature brain, the blueprint of which is determined by the genotype, are “set” differ- ently by external factors and experiences during the maturing period. The lasting effects that occur following exposure to neuro-active chemicals pres- ent in food, the environment or in medicines and drugs, as well as to exter- nal factors like stress, which act on the developing nervous system via endogenous hormonal and neurotransmitter responses, are the result of changes in the cellular make-up, organisation and the synaptic strength of neuronal systems. Pre- and postnatal estrogen exposure modifies later psy- chosexual capacities and gender identity, corticosteroids affect psychomotor behaviour, maternal smoking enhances the incidence of homosexuality amongst female offspring and could be correlated with aggressive behaviour in children of both sexes, neuroleptics impair later learning ability, cocaine Figure 2.1: Prenatal and Life-time Changes of Neuronal Cell Number in the Sexual Dimorphic Nucleus of the Preoptic Area in Male and Female Humans The curves show the fast acquisition of cells in the perinatal period reaching maximal levels only at the age of three to four years for both sexes, and a maturational period with (normal) cell loss until puberty significantly hig- her in girls than in boys, leading to an average 5-fold difference (note the logarithmic scale). It is just a single example of the long lasting brain deve- lopmental period in humans and the influence – in this case sexual hormo- nes – of factors outside of the CNS.

Adapted from Swaab et al. (1992)

impairs later vigilance (detection of actions), and there are more examples to illustrate this interaction (Swaab and Boer 2001). In humans it is difficult to isolate the precise cause of certain characteristics amongst the myriad of stimuli to which a child is exposed. However, as shown above, individual experiences organise the functional capacity of the brain. The knowledge that small changes in the nervous system induced during the perinatal period of brain developmental result in lasting changes to physiology and behaviour, was previously described as behavioural teratology or functional neuroteratology

. Nowadays, this topic also attracts attention in studies on gene-environment interactions as the role of external influences on brain development may be identifiable in the gene expression profiles of nervous structures and may underlie the occurrence or onset of neurological and psychiatric disorders in later life. Above myriad of influences stresses the fact that each brain develops uniquely in its own particular environment, i.e. to its own phenotype. Therefore, the moulding of the nervous system during the development of the brain strongly contributes to the identity and per- sonality of the individual including their capacity to adapt to, or cope with, external challenges.

The influence of external factors on the organisation of the nervous sys- tem continues throughout life through self-reorganisation and changes in number and strength of synaptic connections. This process is called brain plasticity. The window of opportunities for these self-adaptive changes of the nervous system is also set during the period of brain development. For instance, the cognitive abilities of humans depend on the level of plasticity in the nervous systems. A professional activity, for example playing music or learning a second language, will modify the organisation of the brain for the performance of these tasks in everyday life. However, not every nervous sys- tem has the same capabilities in this respect.

The lesson to learn from all this is that intervention in the brain by cell implantation or the genetic modification of cells will modify the organisa- tion of the brain. Therefore, besides the intended therapeutic effects of an intervention on the neural disorder, lasting collateral functional effects can- not be excluded, and must, perhaps, even be expected. This, however, does not mean that restorative neurosurgery should be ruled out completely, although we must consider these factors when assessing the risks and bene- fits of a particular treatment. Ideally, any side effects occurring in patients will be very subtle and will not be obvious in normal daily life.

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This has to be distinguished from classical teratology, i.e., when gross malforma-

tions are visible.

2.3 Concepts of Cell- and Gene-based Neurosurgical Interventions

2.3.1 Historical Outline of Neurotransplantation in Human Beings The loss of neurons in the CNS as the result of degeneration or trauma fol- lowing an accident does not initiate a process of self-repair. Though neuro- genesis takes place at low levels in the adult CNS, the loss of neurons in the above cases is definitive. In addition, in the situation of cell damage, when for instance processes of neurons are transected, neuronal connections can- not autonomically be repaired. The replacement of damaged or degenerated neurons with new ones that will integrate into the broken neuronal system or circuit was successfully undertaken in animals and so became a possibility for human beings too. Immature neurons appeared capable of integrating into the nervous system after implantation, which is not the case for adult neurons. Moreover, damaged neurons that do not restore their connections spontaneously, often maintain the intrinsic capacity to regenerate. Thus, the challenge is to find the conditions to evoke and guide regenerative fiber growth in the damaged brain.

The history of neurotransplantation goes back to 1890, when Thompson attempted to transplant neocortical brain tissue taken from a cat into the brain of dogs. His experiment largely failed, and it took until the 1970s for the work of Das and Altman (1971) and Björklund and Stenevi (1979) to show that only fetal neurons survived brain tissue grafting and that these cells are also able to reconstitute neural circuitry. Thereafter, the grafting of fetal nervous tissue was widely used in animal studies to investigate the processes of brain development. It was also applied as a possible repair strategy in a variety of animals to repair brain damage and neurological dis- orders.

Development towards a human application of these techniques acceler- ated after the observation of Perlow et al. in 1979 that grafting fetal nigral cells into the striatum of substantia nigra-lesioned rats reversed the motor disturbances. These rats were regarded as a partial model for Parkinson’s dis- ease since the gradual loss of dopaminergic neurons of the substantia nigra was seen as the origin of the movement disorders. Models of the disease were developed in primates that better represented the complexity of the disease in humans, with symptoms like tremor, rigidity and bradykinesia (Björk- lund 1992). Subsequent transplantation studies strongly validated the work undertaken in rats (Bakay et al. 1987; Fine et al. 1988; Bankiewicz et al.

1990). These results led to the first clinical studies in 1987, in which human fetal substantia nigra-containing tissue was placed in the dopamine-poor striatum of late stage PD patients (Lindvall et al. 1989; Madrazo et al. 1991).

Hundreds of patients world-wide have received this experimental treatment

since then. The first results were variable and were far from a complete and

persistent recovery from the disease (e.g., Lindvall et al. 1989, Lindvall 1997;

Madrazo et al. 1991; Olanow et al. 1996; Mehta et al. 1997).

The history of neural grafting in human PD can be seen as the test bed for neuron supplementation techniques as a therapeutic intervention in the human brain. It showed cell implantation deep inside the brain to be surgi- cally feasible and proved that restorative neurosurgery using immature neu- rons is possible, at least in principle.

2.3.2 Several Types of Neurotransplants

The above-mentioned application of neural grafts in PD is meant to supple- ment the function of the lost nigro-striatal neurons and to restore the dopaminergic input in the striatum. Neuronal grafting is currently also clin- ically studied in HD patients. In HD it aims at rebuilding the defective stri- ato-frontal pathway.

Immature neurons can either be obtained in one of three ways: i) directly from aborted human embryos and fetuses, ii) indirectly by in vitro prolifer- ation and/or differentiation of stem or germ cells towards the neuronal phe- notype, or iii) through the differentiation of the cell lines of neural precursor cells (“brain-committed cells”). Genuine stem cells, with the potential to dif- ferentiate into neuronal cells (and other organ-specific cell types), can also be collected in three different ways: i) as embryonic stem cells (ESCs) from the blastocyst (pre-implantation embryo), ii) as embryonic germ cells from post-implantation embryos, or as ii) somatic stem cells (SSCs) from organs in late embryonic, fetal, neonatal and adult stages. They can also be obtained from umbilical cord blood. The presence of SSCs in adult organs (often also called adult stem cells) introduces, in principle, the possibility for neural autografts or for the patient to act as their own donor. Brain-committed cells are, for instance, the LBS-neurons (neurons by Layton BioScience Inc.) that originate from a human teratocarcinoma. Teratocarcinomas are tumors of the reproductive organs that are composed of embryonic-like cells that were transformed in the laboratory into fully differentiated, non-dividing neu- rons (Borlongan et al. 1998). Finally, as neurons of non-human mammals can match the functional capabilities of human cells (Isacson and Deacon 1997), implants taken from the brains of pig fetuses are thought to be appli- cable as well.

However, therapeutic approaches that involve something other than

“simply” supplementing neurons are needed when the neuronal functioning

of the anomaly within the brain has an indirect effect on neurons such as in

AD or MS. These other therapeutic techniques include the implantation of a

particular type of glial cells and the implantation of cells that release chemi-

cal compounds to substitute the function of lost neurons (molecular versus

cellular replacement), or release compounds that can stop, prevent or coun-

teract the degeneration or malfunction of diseased neurons (molecular

treatment) (see figure 2.2). Such cells do not need to be neural cells but can

also be non-neural cells isolated from other human organs, like fibroblast taken from the skin. Some of these approaches are already in the first phase of clinical evaluation. In the case of MS, for instance, glial cells of the oligo- Figure 2.2: Neurotransplantaion as a Kind of Restorative Neurosurgery Comes with a Variety of Possibilities for Various Types of Defects in the CNS

When neurons are lost in neurodegenerative diseases, or following a brain trauma, immature neurons, either dissected from young stages of the human (or pig) brain or manifactured in the laboratory from relevant sources (e.g stem cells, certain teratotomas, germline cells, etc.), can, upon grafting, replace the loss through a process of maturation and integration in the affected host nervous circuit over a period of several months. Embryonic and fetal brain tissue can either be placed as tissue fragments or as cell suspen- sions, but have to be dissected at the proper immature stage of donor devel- opment for the neurons to be replenished. Similarly, immature neurons can replenish dys- or malfunctioning neurons, whereas glial cell loss in the nerv- ous system (“supporting” brain cells present 10-fold the number of neurons) can also be supplemented after these cells are proliferated in the laboratory to the volume needed. However, instead of cell replenishment, cells can also be implanted for their specific action on damaged, degenerating or dysfunction- ing brain areas by releasing supporting or growth-stimulating proteins (mol- ecular supplementation; cells as chronic proteineous drug delivery prepara- tions). These cells do not necessarily have to be neural cells. They can even be animal-derived cells, especially when encapsulated by semi-permeable mem- branes that avoid tissue immunorejection. The growth of cells in the labora- tory is often accompanied with genetic modification either to direct proper neuronal differentiation or to equip them with a gene for the overexpression of a therapeutic compound prior neurotransplantation.

The use of cultured cells allows auto-transplantation as well. Cells with-

drawn or dissected from the patient him-/herself are used for laboratory

growth, thereby again preventing the need for immune-suppression ther-

apy when grafted.

dendocytic type are the cells to be supplemented and only implants with cells that have been grown and purified in vitro are used.

2.3.3 The Practice of Clinical Neurotransplantation

The survival and integration of transplanted nerve cells depends on their plastic growth capacities at the stage of maturation in which they have not yet fully developed their complex neurite connections and bio-electrical interactions. In the brain developmental period each type of nerve cells has its own time window of “birth” and its own pace of maturation. The trans- plantation of entire brain sub-regions might thus easily result in one cell type surviving and another, more mature, cell type failing to do so. Conse- quently, a large brain part may survive transplantation as a tissue mass, but it can or will not easily develop its normal organisation in the recipient brain, nor develop the proper connections with, or within, the damaged neuronal systems of the brain. In other words, neurotransplantation strategies in human patients add new cells of particular types (cell suspensions), or place fragments of immature brain structures (minced tissue) but cannot aim to replace entire brain structures that are lost due to severe damage or trauma, as occurs, for example, in the case of heart and kidney transplantation.

Nothing will be removed from the CNS for replacement, and parts cannot be replaced like a module of a defective computer. Neurotransplantation should therefore not be described as brain transplantation, but only as brain cell or brain tissue grafting.

In practice, neurotransplantation in defective areas of the human brain consists in the precisely directed injection of microliter quantities of suspen- sions of nerve cells or tissue fragments prepared from defined areas of fetal brain known to contain the needed cell type in an immature state or prepared from cells specially cultured and modified for it in the laboratory. The injected mass is about 100,000 times smaller than the volume of the adult brain (approximately 1.5 liter). This type of intervention requires surgical precision and accuracy, but it is not an extremely severe, physically invasive operation on the patient. Transplanted nerve cells have to mature and integrate for proper restoration of the lost brain function to occur. The possible therapeutic effects of neurotransplantation are, therefore, never immediate, but develop over a period of several months, not unlike the time frame of brain cell maturation in the intrauterine fetal stages (Isacson and Deacon 1997).

2.3.4 Direct and Indirect Gene Transfer in the Brain

Modification of the gene expression of cells in a living organ became possi-

ble with the development of viral vectors – genetically modified viruses that

infect a cell, but cannot replicate nor evoke its disease effects – that can

deliver a therapeutic gene in a target cell. This form of gene transfer aims to

i) restore protein expression in a hereditary failing molecular or cellular

process, ii) compensate for the loss of particular protein expression (in

degenerative diseases) or iii) (over)express proteins that have symptom- relieving or restorative effects as a locally delivered drug (as externally deliv- ered non-proteineous drug can have). Gene transfer can additionally be used to iv) block the endogenous expression of proteins which cause the symp- toms of a disease or v) frustrate tissue self-repair following trauma making use of siRNA (small interfering RNA) technology

. In the nervous system two approaches can be distinguished for the (over)expression of proteins: in vivo (direct) and ex vivo (indirect) gene transfer (Kaplitt and During 2006).

The first approach involves the injection of viral vectors carrying the gene of interest directly into the brain tissue of the patient. In the second approach the cells to be implanted are genetically modified in vitro, and then har- vested for implantation surgery (figure 2.3). For the latter case also non-viral vector transduction methods are available in which the cells can be cultured as single cells.

The potential applications of gene transfer in restorative neurosurgery are manifold. In vivo transduction could equip degenerating neuronal or glial cells with properties to survive damage, to restore a lost function, or to release compounds that serve these purposes in adjacent cells. In vivo gene transfer for neurotrophic factors like nerve growth factor (NGF), brain- derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) and neurotrophin-3 (NT-3) in animal models for neurodegenerative diseases and neurotrauma have shown themselves to be very potent in increasing cell survival and/or promoting axon sprouting. Viral vector-mediated gene transfer has been applied exper- imentally in human beings for diseases outside the nervous system and, recently in the CNS as a myriad of neurological disorders may also be treat- able according to the results of animal experimentation (Tuszynski 2002;

Kaplitt and During 2006). Viral vectors not only need to be able to infect a post-mitotic neuron, but it should also have no toxic or immunological effects on CNS tissues and should provide long term, and preferably control- lable, gene expression of the therapeutic protein that should be locally effec- tive without affecting intact neighbouring neural systems too much.

Currently lentiviral (LV) vectors and adeno-associated viral (AAV) vec- tors are vectors that efficaciously and directly transduce the CNS tissue with- out direct or short term toxicity for neuronal cells. Whether it is safe for use in the human brain in the long term still has to be established. Clinical trials with both ex vivo gene transfer and direct gene transfer are currently being performed in PD and AD patients (see below).

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Small interfering RNAs (siRNAs; also called “short interfering RNAs”) are a class of 20–25 nucleotide-long RNA molecules that interfere with gene expression.

They are naturally produced as part of the RNA interference (RNAi) pathway, but

can be designed and artifically applied to inhibit endogenous expression of pro-

teins from a particular gene.

Figure 2.3: The Principles of Gene Transfer for therapy

Either a neuron or a non-neuronal cell can be modified by the insertion of a gene (transduction) for the production of survival-enhancing and growth-stimulating factors or of factors that mimic or enhance neuronal functions in the impaired or damaged CNS. Nowadays, the most efficient and safest way to modify neurons is the use of viral vectors. Viral vectors are viruses that are (re)constructed so that they can infect a cell but do not have the capacity to multiply following infection. This is achieved by creating viruses that do not contain the DNA for their reproduction but instead con- tain the DNA of the therapeutic gene, which can be delivered to the target cell nucleus to initiate the transcriptional and translational machinery for the synthesis of the therapeutic protein.

Viral vector-mediated transduction can be applied by direct injection into

the CNS or it can be used in combination with neurotransplantation (ex

vivo transduction). When the source cells used to grow transplants are

obtained from the patient her-/himself, auto-implantation is also possible

in order to prevent the immune-suppression treatment required following

the allografting of cells from human donors or animal sources.

2.4 Survey of Current Experimental Human Applications of Restorative Neurosurgery

Clinical application of cell implantation and gene transfer in human brain disorders has not reached the level of therapy. All treatments still are in the experimental phase as the beneficial functional outcomes are variable, unpredictable or not present at all. The following section surveys briefly the achievements in this area to date.

2.4.1 Parkinson’s Disease

PD is primarily caused by the slow loss of dopaminergic neurons in the sub- stantia nigra so that their dopamine transmitter function in the striatum eventually disappears. PD is generally age-specific: approximately 1% of the population over age 60 develops the disease. An appropriate dopaminergic signal is vital for a smooth, coordinated function of the body’s muscles and movement. As soon as approximately 80% of the dopamine-producing cells are lost, the symptoms of Parkinson’s disease appear. The key signs of PD are tremor, slowness of movement, rigidity and loss of balance. Other signs of Parkinson’s disease may include small, cramped handwriting, stiff facial expression, a shuffling walk, muffled speech and depression. Current phar- macological treatments with dopamine agonists and dopamine precursors reduce the symptoms in the early stages of the disease. However, with progress of the nigral degeneration, these drugs cease to be effective.

Dopamine cell supplementation began with open trials of striatal place- ment of the patient’s own dopamine-producing adrenal medulla tissue (Backlund et al. 1985; Madrazo et al. 1987). This tissue was used experimen- tally as an alternative source of dopamine in order to circumvent the ethical problems following the use of human fetal brain tissue obtained from elec- tive abortions (Boer 1996). The outcomes of this and later studies by other groups were disappointing and must be considered to have largely failed: not enough of the transplanted tissue survived, amelioration of the motor dis- turbances was absent or minor, and no relationship existed between dopaminergic cell survival and behavioural response. Other approaches of bypassing the use of human embryonic tissue have been tried, including cells obtained from the patient’s own stellate ganglion but only modest anti- parkinsonian effects are reported in a small number of patients (Itakura et al. 1997). As a corollary of the above clinical results, as well as the signifi- cantly better functional effects of immature tissue transplants in the case of parkinsonian rats and monkeys, a move towards the use of human fetal dopaminergic neurons in patients was inevitable (Boer 1999).

Intracerebral transplantation of human fetal dopaminergic neuron-con-

taining mesencephalic tissue fragments, or cell suspensions thereof, obtained

from the remains of legally induced abortions, were placed in the dopamine-

depleted caudate-putamen complex of late stage PD patients. So far, more

than 300 patients with PD have undergone this allograft surgery, but under different conditions of donor tissue treatment, graft placement, surgical approach and pre- and post-grafting treatment and symptom evaluation.

Months after the implantation surgery several clinical centres observed con- sistent and clinically meaningful benefits in small groups of patients in open trials using a relatively strict common protocol of pre- and post-surgery evaluation of graft survival and disease symptoms (Peschanski et al. 1994;

Defer et al. 1996; Levivier et al. 1997; Mendez et al. 2002). Others, however, reported more variable or negative results (Freed et al. 1992; Lopez-Lozano et al. 1997). The benefits on the Unified Parkinson’s Disease Rating Scale (UPDRS) often go hand in hand with dopaminergic cell survival as meas- ured by fluoro-dopa PET scanning, which indicates graft survival.

Recently, the results of randomised double-blind sham surgery-con- trolled neurotransplantation studies in PD were published (sham surgery performed as a hole drilled in the outer layer of the skull bone but without penetration of a canula into the brain) (Freed et al. 2001, Olanow et al.

2003). At the outset, the design of such studies was criticised with respect to the fact that this large-scale study, including ~20 patients in each group, was performed at too early a stage, i.e., when optimal methods for tissue pro- curement, graft preparation and implantation had not yet been established (Widner 1994). The studies did, however, demonstrate that there was no lasting placebo effect, and that anti-parkinson effects were found primarily in the younger group of patients (Freed et al. 2001). Moreover, several patients in the treatment group developed abnormal involuntary move- ments and these movements were regarded as major side effects of this study.

The modest improvement in neurological rating scores, only partly compa-

rable with other studies (Isacson et al. 2001), and the occurrence of dyskine-

sias aroused widespread scientific interest and debate about the future of cell

replacement therapies in PD (Brundin et al. 2001; Dunnett et al. 2001; Isac-

son et al. 2001). The fact that the study was double-blind and sham-con-

trolled eased the initial methodological criticism and led the media to take

these results as sound evidence that the technique of neural tissue transplan-

tation in general was faulty and ineffective (Vogel 2001). This interpretation,

however, is erroneous, as the net effects are dependent on the particular

technique used (Björklund 2005). What was predicted by Widner and Defer

(1999) became true: the results of a suboptimal grafting procedure chal-

lenged the therapeutic value of cell therapy in PD. Journalists called the

results a failure, thereby harming the field that tries to develop novel cell

replacement therapies in brain diseases (Dunnett et al. 2001). However, the

field of experimental clinical neurotransplantation agreed that dopaminer-

gic cell implantation in PD cannot be recommended as, or even be called, a

therapy (Polgar et al. 2003). Further improvement of the technique is needed

and the cause of the dopaminergic graft-related dyskinesias needs to be

unraveled.

In addition to cellular therapies in PD, phase I studies are currently also being performed with AAV vector-mediated gene transfer, based on a series of successful studies with in vivo and ex vivo AAV and LV vector-mediated gene transfer in PD animal models (Raymon et al. 1997; Freese 1999; Kor- dower et al. 2000; Shen et al. 2000; Le and Frim 2002). One trial tries to mimic the results of deep brain stimulation (DBS) in the subthalamic nucleus (STN) of the brain. DBS is shown to be an effective method to treat many PD patients in the late stages of the disease when L-dopa medication starts to fail. The application of AAV-GAD vectors (containing the gene for glutamic acid decarboxylase [GAD], the enzyme synthesising the major inhibitory neurotransmitter gamma amino butyric acid [GABA] and upon overexpression causing a chronic release of GABA) in the animal STN results in similar result as electrical stimulation (During et al. 1998; 2001). Accord- ing to the interim clinical findings (Feigin et al. 2005), AAV-GAD treatment in the STN appears to be safe and well-tolerated in advanced Parkinson’s dis- ease, with no evidence of adverse effects or immunologic reaction. One year after treatment, patients exhibited a 27% statistically significant improve- ment in motor function on the side of their body corresponding to the treated part of the brain, with no improvement for the untreated side. A sec- ond phase I clinical study uses AAV-AADC, a vector that introduces the gene for L-amino acid decarboxylase (AADC) in the striatum of PD patients (http://www.avigen.com, accessed on December 7

, 2006). This enzyme catalyses the synthesis of dopamine, and is known to decrease with progres- sion of PD. In parkinsonian monkeys, the vector has been effectively applied (Bankiewicz et al. 2000; Sanftner et al. 2005) and may be of continuing clin- ical benefit (Bankiewicz et al. 2006).

2.4.2 Huntington’s Disease

HD is a rare autosomal dominant neurodegenerative disease that causes devastating disorders. It affects principally people above the age of forty. The disease inalterably proceeds towards a multi-faceted cognitive deterioration, motor disorder-associated chorea and bradykinesia as well as psychiatric disturbances such as depression and irritability. The clinical symptoms – at least in the early stages of the disease – are related primarily with a hypo- functioning and a degeneration of the medium spiny GABAergic neurons in the striatum. In the later stages of the disease, cortical and sub-cortical struc- tures, anatomically connected with the striatum, become affected too. The disease is fatal within 15 to 20 years of its onset in most patients (Bird and Coyle 1986) and has no cure or any effective treatment. Besides the search for therapeutic agents like neurotrophic factors which act against the molec- ular mechanisms of neurodegeneration in HD, therapeutic research also focuses on GABAergic nerve cell supplementation therapy.

Intrastriatal implantation of (striatal) fetal ganglionic eminence tissue

was able to reverse a large number of the motor and cognitive deficits

brought about by striatal lesions of various kinds in animal affected by HD (cf. Peschanski et al. 1995). Several indicators suggest that implanted neu- rons do mature normally, are mainly GABAergic and express both the expected corresponding neuropeptides (substance P, met-enkephalin, somatostatin or neuropeptide Y) and the dopaminergic and muscarinic receptors. Host afferent axons both grow into the grafts and connect to grafted neurons (cf. Wictorin 1992), and functional reconnection of grafted GABAergic cells to the experimentally denervated target neurons of the globus pallidus also develops. Grafted neurons do not reach more remote projection zones such as the substantia nigra, pars reticulata, but the globus pallidus is by far the most important projection zone of striatal neurons in primates and in humans. Behavioural analysis of grafted animals to a large extent confirms the rewiring of cortical output circuits in which striatal neu- rons normally act as first relay cells (Dunnett et al. 1988; Kendall et al. 1998;

Palfi et al. 1998; Hantraye et al. 1990).

The converging evidence in animal studies, outlined above, has led to tri- als of intracerebral grafting in patients with HD. Except for the study carried out by Hauser et al. (2002), all studies involved patients at an early stage of the disease. The safety and feasibility of the grafting procedures appeared almost unquestioned in all studies (Kopyov et al. 1998; Bachoud-Lévi et al.

2000b; Fink et al. 2000; Rosser et al. 2002) except in the Hauser et al. (2002) study where patients at a more advanced stage of the disease, and patients with a history of neurological problems, were included and some subjects developed subdural hemorrhages or required surgical drainage. The latter may indicate that patients at an advanced stage of the disease are particularly sensitive to medical interventions. No noticeable side effects were reported in the other studies except for difficulties encountered in obtaining a good compliance of patients for drug treatment and, in particular, for immuno- suppressive drugs (Bachoud-Lévi et al. 2000b; Rosser et al. 2002). An autopsy in one patient, who died of causes unrelated to the transplant 18 months after surgery, revealed the presence of a large graft that contained a large number of neurons phenotypically similar to GABAergic medium-spiny striatal neurons (Freeman et al. 2000). Moreover, the grafted cells did not exhibit any signs of the disease, e.g. nuclear inclusions, in contrast with the host neurons in the surrounding striatum.

Conclusive clinical benefits so far have only been shown in the Créteil clinical trial by Bachoud-Lévi et al. (2000b; 2002). The other study, whose clinical data has now been published (Tampa trial; Hauser et al. 2002), was inconclusive. This was possibly due to the type of patients included or the fact that it allowed too short a follow-up time (Peschanski and Dunnett 2002). In Créteil, an improvement in motor, cognitive and functional abili- ties became apparent only at about twelve months in three of five HD patients, and remained so in the subsequent two years (Bachoud-Lévi et al.

2000b). These clinical results matched with the reduction of both the striatal

and the frontal hypometabolism as measured by positron emission tomog- raphy using

F-fluorodeoxyglucose (Gaura et al. 2004). In a fourth patient, this improvement was transient, starting around nine to ten months after a first right-side unilateral graft, and lasted up to five months after a second left-sided graft. In this patient, the secondary loss of all improvements coin- cided with the disappearance of the grafted tissue as evaluated with MRI (Bachoud-Lévi et al. 2002), indicating a link between graft survival and clin- ical benefits. In the fifth patient, the graft was never active for reasons that remain unknown, and MRI scans still shows declining signals for the striatal metabolic activity (Bachoud-Lévi et al. 2002). Therefore, despite the absence of a control group, the coincidence of results acquired in various domains (clinical, images, electrophysiology) and analysed blindly, strongly point to the efficacy of neurografting. The positive treatment result in a very small population of HD patients obtained in a single centre trial initiated in 2001, initiated a large, controlled randomised trial on 60 patients at the early stages of HD in France and Belgium (Multicenter Intracerebral Grafting in HD, MIG-HD). For control purposes, and to avoid the use of sham surgery, 30 patients randomly received transplants either after 13–14 months or after 33–34 months, with a follow-up of all patients towards 52 months. Cur- rently, this strategy is being replicated in a separate study in which the results of grafts conducted in Belgium, Germany, Switzerland and Italy will be com- pared with a large cohort of non-treated patients in the UK. Therefore, the efficacy of fetal neural grafts as putative therapy for HD will be fully known in the next three to five years.

The sustainability of the positive effect resulting from grafts is currently being assessed in the patients from the Créteil‘s pilot study (Bachoud-Lévi et al. 2006). The gene defect is still present and the patients‘ condition is expected to deteriorate at some point in the future. This secondary deterio- ration has appeared heterogeneous so far, starting at 4–5 years in the case of motor symptoms and after 6 years in the case of cognitive functions. Thus, the potential therapeutic effects of fetal striatal grafts possibly fade away due to a process of remission. This indicates that a neuroprotective treatment of the graft is needed as an unavoidable complement to the initial surgery.

However, the graft will remain the only therapy able to restore lost functions and, therefore, will be indicated in patients exhibiting the symptoms of HD.

A number of experimental studies conducted on animals affected by HD

striatal lesions have demonstrated that various neurotrophic factors can pro-

vide neuroprotection. Among these factors CNTF appeared to offer the most

effective protection. However, the short half life of the CNTF in plasma, its

inability to cross the blood brain barrier and its severe side effects (inflam-

mation, cachexia) in a phase I/III clinical trial in patients with ALS (Cedar-

baum et al. 1995), precludes its systemic administration. Following the posi-

tive results of striatal protection in rats and non-human primates using

CNTF-delivering mini-pumps (Anderson et al. 1996) or gene therapy

approaches (Emerich et al. 1996; 1997; Mittoux et al. 2000; 2002), an intra- ventricular implant of encapsulated CNTF-producing cells was chosen for a phase I trial (Bachoud-Lévi et al. 2000a). Cells were taken from a baby ham- ster cell line engineered to synthesise and release large amounts of CNTF which were subsequently introduced into semi-permeable tubes with pores i) permitting CNTF and all nutrients to cross the membrane, and ii) exclud- ing larger proteins (e.g. antibodies) and cell processes to traverse. The cell encapsulation method has the advantage that in the clinical situation it immuno-isolates the cells, whereas removal of the device can stop the treat- ment whenever needed. The capsule was inserted into the lateral ventricle of six HD patients using stereotactic neurosurgery and was retrieved and exchanged every six months during a two year period. Little, if any,

F-fluo- rdeoxyglucose-determined metabolic change was observed in the ipsilateral striatum, but significant recovery of normal electrophysiological values was associated with active CNTF-releasing tubes in three patients (Bloch et al.

2004). There were no adverse effects related to the procedure. However, sec- ondary adverse effects (mainly depression) related to the interruption of the procedure were observed a few months after the extraction of the last tube, showing the symbolic and emotional aspect of such therapy.

2.4.3 Alzheimer’s Disease

AD is a neurodegenerative disease associated with the formation of tangles and plaques in the brain, resulting in neuronal atrophy leading first to mild forgetfulness (which can be confused with age-related memory change) and an inability to solve simple mathematical problems and followed later by severe cognitive deficits and problems in speaking, understanding, reading and writing. In the final stages of the disease, patients often exhibit anxious- ness or aggressiveness and become in need of total care. The precise cellular or molecular origin of the disease is not known, so that there is no clearly definable “point of attack” at which to fight the cause of the disease nor its progress. Yet certain symptoms can be traced back to changes in particular brain nuclei. For instance, cholinergic neurons of the basal forebrain atrophy and die in the brains of those affected with AD. This process has been corre- lated with attention deficits and an overall cognitive decline. As the applica- tion of NGF in this area has shown to protect cholinergic cell loss following their axotomy (cf. Lad et al. 2003), the chronic delivery of NGF in the human basal forebrain to reduce, or prevent, the loss of cholinergic nerve cells could possibly result in the relief of these symptoms (Tuszynski 2002).

In order to investigate the effect of NGF in AD patients, local application

is needed as infusion of NGF in the ventricles of the brain results in intoler-

able side effects. For instance, rats and monkeys undergoing cholinergic cell

rescue procedures using NGF lost their appetite leading to severe weight

losses, and this was also observed in a clinical trial of NGF infusion involving

three AD patients (which had to be stopped because of painful side effects

were experienced by the patients; Eriksdotter Jonhagen et al. [1998]). These negative effects were not observed in subsequent studies in which autolo- gous NGF-secreting cells were implanted into the cholinergic basal forebrain of aged monkeys in which a substantial reversal of age-related neuronal atro- phy was achieved (Tuszynski et al. 1998; Tuszynski and Blesch 2004). This had led to a phase I clinical trial of ex vivo NGF gene delivery through the implantation of transduced fibroblasts isolated from the skin of the AD patient, grown and transduced ex vivo (Tuszynski et al. 2005). In this study, after a mean follow-up of 22 months in six AD subjects, no long-term adverse effects resulting from the NGF occurred. Preliminary outcomes showed the Mini-Mental Status Examination and Alzheimer Disease Assess- ment Scale-Cognitive subcomponent to be improved suggesting cognitive decline have decelerated. Serial PET scans showed significant increases in cortical

F-fluorodeoxyglucose after treatment, indicating a return of brain activity at pre-disease stages. The brain autopsy from one subject suggested robust neurite growth responses to NGF.

2.4.4 Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) causes the progressive degeneration of motoneurons of the CNS. If the motor neurons die, the ability of the brain to initiate and control muscle movement is lost. The course of the symptoms starts with muscle weakness in one or more muscles of the hands, arms, legs or of the muscles involved in speech, swallowing or breathing. This then develops into twitching and a cramping of muscles, impairment in the use of the arms and legs (paralysis), “thick speech” and, in advanced stages, diffi- culty in breathing and swallowing, eventually leading to death. Yet, for the vast majority of ALS patients, their minds remain unaffected throughout.

Currently there is no treatment for this disorder starting with loss of func- tion of the motoneurons in the spinal cord.

Ciliary neurotrophic factor (CNTF) has been shown to protect motoneu-

rons from deterioration. Thus, subsequent patient studies with this peptide

were initiated. Systemic delivery of hCNTF in ALS patients, however, had no

beneficial effect on the primary (limb strength and pulmonary function) or

secondary end points (individual function tests and activities-of-daily-

living outcome measures and survival; Miller et al. 1996), but has been frus-

trated by peripheral side effects, as well as the molecule’s short half life, and

its inability to cross the blood-brain barrier. Aebischer and his collaborators

(Sagot et al. 1995; Tan et al. 1996) have conducted experiments in mice with

symptoms of ALS which showed that encapsulated baby hamster kidney

cells, genetically engineered to make CNTF and placed intracerebroventri-

cally or intrathecally, also reduced the degeneration of these neurons. Trials

in human ALS patients in 1996, using the same procedure described above in

the treatment of HD but involving the implantation of the CNTF-releasing

tube intrathecally in the lumbar CNS (Aebischer et al. 1996; Zurn et al.

2000), showed no evidence that the CNTF alleviated motor neuron deterio- ration (P. Aebischer, personal communication). One of the reasons for this could be that insufficient amounts of CNTF were released or that the inter- vention was too late to be of use (Schorr et al. 1996).

Huang et al. reported significant improvements in ALS patients after the implantation of human fetal olfactory ensheathing glia cells in the motor cortex of the brain at an international conference in 2004. However, the rationale of this surgery, advertised as a therapy to both prolong the life span and improve the quality of life of patients, is not based on animal experi- mentation, and the study has still not been published in a peer-reviewed journal in which the methods they employed would be evaluated in detail.

2.4.5 Multiple Sclerosis

Multiple sclerosis (MS) is an unpredictable, chronic disease of the CNS, whose symptoms can range from the relatively benign to the somewhat and potentially devastating. Pathologically, MS is characterised by the presence of areas of demyelination and predominant T-cell perivascular inflamma- tion in the brain white matter, which disrupts efficient communication between the brain and other parts of the body. MS is believed to be an autoimmune disease that attacks the nerve-insulating myelin. Common symptoms of MS include fatigue, weakness, spasticity, balance problems, bladder and bowel problems, numbness, loss of vision, tremors and depres- sion. Symptoms are determined by the location of the lesion and thus not all symptoms affect all MS patients. Symptoms may be continuous or may be sporadic. These periods of remission may be complete, leaving no residual damage or leaving only partial permanent impairment. A variety of medica- tion can be used to treat the disease symptomatically, but there is, as yet, no cure for the demyelination in MS. New therapies, therefore, need to aim at reducing specific autoimmune responses and to assist in remyelination. It is the latter goal in which neurotransplantation may have potential.

Animal studies have shown remyelination processes following cellular

therapies in experimental demyelination (Kocsis et al. 2002). The use of

Schwann cells, glial cells that normally insulate axons in the PNS, were found

to remyelinate fibers in the CNS of rats and reinstate message transmission

(Kohama et al. 2001; Bachelin et al. 2005). In a 2001 pilot study Tomothy

Vollmer and co-workers (http://www.myelin.org/schwannupdate.htm,

accessed on December 7

, 2006) transplanted autologous Schwann cells in

three patients with MS and found that the technique was safe. Further stud-

ies are needed to determine whether the cells can also repair myelin and aid

functional improvement in patients. Other cells for remyelination are olfac-

tory ensheathing cells that inhabit the nose but can also make myelin

(Franklin et al. 1996; Lakatos et al. 2003) and neural stem cells, which assist

to stimulate remyelination by endogenous oligodendrocyte precursor cells

or mature themselves into oligodendrocytes and subsequently produce

myelin in the CNS (Totoiu et al. 2004; Copray et al. 2006). In one recent study scientists found that in mice with an MS-like disease, transplants of stem cells travelled to multiple areas of damage and matured into myelin- forming cells. Animals undergoing such transplantation showed a decrease in myelin damage and nerve fiber destruction. Some animals also regained lost movement in their legs or tails (Pluchino et al. 2003; Pluchino and Matino 2005).

A large clinical trial using autologous SSCs from bone marrow or blood as peripheral implants not as brain implants, combined with high-dose immunosuppression, revealed slight neurological improvements in 21% of the MS patients and a stabilisation of the clinical condition in approximately 70% of the patients trialed by completely abrogating the inflammatory process in the brain as evidenced in magnetic resonance imaging (Fassas et al. 2002). However, the procedure is associated with a transplant-related mortality risk of around 3% to 8%. Therefore, it cannot be recommended for the treatment of a chronic, non-lethal disease like MS. However, the sys- temic or peripheral approach of cellular treatment has the advantage that the skull need not be opened up for surgery. On the other hand, a direct approach of the MS lesion area for any type of therapy may be more effective and reduce the chances of side effects due to maladaptive myelination in uninjured parts of the brain.

2.4.6 Stroke

Brain stroke occurs when blood supply to, or within, the brain region stops.

A stroke can occur anywhere in the CNS and is caused either by a cerebral

infarction, as a result of a blocked artery (ischemic stroke) or by an intracra-

nial or cerebral haemorrhage as a result of weak arteries or an aneurism in

the brain that ruptures (haemorrhagic stroke). The symptoms a stroke vic-

tim experiences depend on which areas of the brain are involved and can

include, amongst other symptoms, an abrupt loss of vision, coordination,

sensation, speech, paralysis and loss of consciousness. Brain stem strokes are

especially devastating and life threatening because they can disrupt the

involuntary vegetative functions essential to life. When blood supply is

blocked, brain cells die as they are deprived from oxygen (ischemia), and

they start to release toxic chemicals that threaten surrounding tissue (the

ischemic penumbra). In ischemic stroke, the acute goal is to restore blood

flow to the area and to prevent cell death in the penumbra. Thrombolytic

drugs are nowadays applied as a “blood clot-buster” to restore blood flow

(Pulsinelli et al. 1997). A variety of cytoprotective agents can be used in the

post-acute phase for up to six hours (Endres et al. 1998), but their effective-

ness is poor and the treatment window limits its application to only a small

number of patients. In hemorrhagic stroke, however, thrombolytic drugs

would actually have a detremental affect (Schellinger et al. 1997). If sponta-

neous clotting does not occur and hematoma increases in size, a rapid neu-

rosurgical intervention may be needed to stop bleeding. Depending on the site, the duration and the severity of the blocked or hampered blood supply, the patient usually recovers, but often a lasting defect remains due to a loss of brain tissue, which is also visible in brain scans. The brain can compensate for this damage to some extent. Some neurons may only have been tem- porarily damaged, not killed, and the plasticity of the brain allows it to reor- ganise neuronal networks so that other parallel brain areas can take over functions stimulated by physical, occupational, and speech and audiology rehabilitation programmes. However, large infarctions or chronic cases of small strokes require tissue repair. The ischemic penumbra is the target area for both restoration and the prevention of further neuronal degeneration.

The possibilities of neurotransplantation guided experimental studies in rodent early stroke models to either replace the lost neurons or place cells as a source of trophic factors to enhance plasticity phenomena for recovery of function. It proved to be effective in many studies (cf. Abe 2000; Nishino and Borlongan 2000). Fetal neurons (Netto et al. 1993) and cultured LBS neu- rons, grown and differentiated from a malignant human testicular carci- noma (Borlongan et al. 1998) were found to integrate with existing neurons in the stroke affected area in rats and to correct cognitive and motor skill problems. In addition, human neuroprogenitor cells (Kelly et al. 2004), human bone marrow stem cells (Zhao et al. 2002) appear to exhibit a similar effect, and these cells differentiate themselves to resemble the neighbouring cells in the site of the lesion. However, the observed functional improve- ments are possibly mediated more by proteins secreted from the implanted cells than by cell supplementation since the integration of implanted cells in the host brain is limited. Thus, an upregulated host brain plasticity may be the underlying mechanism. This trophic mechanism is also assumed to take place following implantation of human umbilical cord blood cells (Ven- drame et al. 2005) or porcine choroid plexus tissue (Borlongan et al. 2004), but concurrent angiogenesis may occur as well (Jiang et al. 2005).

The early positive and encouraging results with the LBS neurons in rat

stroke models has led to clinical trials in patients with chronic motor defects

resulting from an ischemic stroke. The cells were implanted with multiple

injections around the area of the brain lesion in patients whose stroke

occurred six months to six years previously and who had a fixed motor

deficit that had remained stable for at least two months in order to evaluate

any possible improvements resulting from the procedure. This phase I trial,

including twelve patients, showed no adverse cell-related serologic or imag-

ing-defined effects up to 18 months after surgery. There was also evidence of

improved metabolism at the implant site in seven patients and some

improvement on the European stroke scale score in six patients (Kondziolka

et al. 2000). A positive correlation was found between glucose metabolic

activity in the stroke area and motor performance (Meltzer et al. 2001) and

cognitive function improved for those patients treated for basal ganglia

stroke after six months (Stilley et al. 2004). The subsequent phase II trial with LBS neurons using pre- and postoperative, observer-blind evaluations and control patients for comparison, revealed no significant benefit in motor function, although several patients noted measurable improvements of their functional defects in daily life compared to pre-surgery state (Kondziolka et al. 2005). So again neuron implantation is feasible in patients with motor area infarction, but a genuine and reproducable therapy was not reached. Other types of transplants, such as fetal porcine cells (Savitz et al.

2005) and cell suspensions from immature human nervous and hemopoietic tissues (Rabinovich et al. 2005), were also applied in small pilot studies with partial success. However, these studies were not based on any preclinical ani- mal studies.

2.4.7 Epilepsy

The hallmark of epilepsy is the occurrence of usually unpredictable, sponta- neous seizures in the brain. These seizures are an event with a particular focus in the CNS; the cause is not precisely understood. It is either a symp- tom of specific congenital diseases or acquired following injury to the brain from sclerosis, tumors, abscesses, strokes or gliosis. Focal epilepsies can often be controlled by drugs that favour inhibitory over excitatory neurotransmis- sion. Seizure activity, however, persists in approximately 35% of the patients taking these anti-epileptic drugs (Devinsky 1999). Medically intractable epilepsy, in cases of an identifiable epileptic focus, may be treatable through lesion surgery. Even so, in a number of patients surgery fails to control the seizures, and many patients cannot be surgically helped because of the (often extremely high) risk of losing important brain functions such as speech and motor control. Among the various new treatment techniques under investi- gation (Rosenfeld 2002), neurotransplantation and gene transfer were recently proposed after breakthroughs in the treatment of epileptic animal models (Freeman 2000).

Cells engineered to release GABA, the major inhibitory transmitter, or

adenosine, known to suppress seizure activity, have been applied successfully

as anticonvulsant treatments in rats experiencing chronic seizures (Löscher

et al. 1998; Gernert et al. 2002). GABA-releasing cells are conditionally

immortalised neurons genetically modified to over-express the GABA-syn-

thetising enzyme GAD under the control of tetracycline. These cells, when

placed intraparenchymally in the brain of animals experiencing sponta-

neous seizures, brought about a reduction in the number of spontaneous

seizures (Thompson and Suchomelova 2004; Thompson 2005). The adeno-

sine-releasing cells were modified by the genetic inactivation of adenosine

kinase or aminase enzymes that normally break down adenosine. Encapsu-

lated in semi-permeable membranes (see above in the section on HD), these

cells prevent kindling-induced epilepsy in rats when placed intracerebroven-

tricularly (Huber et al. 2001; Guttinger et al. 2005a).

The first human pilot study of the implantation of GABA-producing cells was performed in epilepsy patients who failed to respond to conventional epilepsy medication, and who are candidates for the surgical removal of a portion of the brain in order to control seizures (D. Schomer et al.; commu- nicated at the 58

Annual meeting of the American Epilepsy Society, New Orleans 2004). These cells were fetal porcine neurons and the study aimed primarily to look at cell survival, host reaction, and clinical side effects. An ability to control seizures was reported in two out of three patients in this unblinded study. However, during the subsequent epilepsy lesion surgery, no implanted tissue was detected. The study was stopped as a result of this and also because of the concern about safety of porcine xenografting (see below).

Currently, fundamental research is moving towards the option of using (human) neural stem cells as they differentiate into GABAergic neurons (Chu et al. 2004) following brain implantation. Moreover, they can be genet- ically modified so that they release seizure-reducing molecules (Guttinger et al. 2005b).

If cell implants can have an anti-epileptic effect through the release of seizure-reducing compounds, direct genetic modification of the cells in or around the epileptic focus would be an obvious alternative. The generation of AAV and LV viral vectors, which are capable of stable transduction of neurons, is an example of this type of strategy. Indeed, animal studies showed that an overexpression of galanin (Haberman et al. 2003; Lin et al.

2003) and neuropeptide Y (NPY) (Richichi et al. 2004; Noe et al. 2005) revealed significant anticonvulsant and anti-epileptic effects. Phase I studies with AAV-NPY treatment in intractable epilepsy are reported to be on the way (Neurologix; http://www.neurologix.net, accessed on December 7

, 2006).

2.5 When is a Brain Disorder Eligible for Cellular or Molecular Surgery?

The history of cellular intervention in the human brain started with the

autologous implantation of adrenal medulla tissue fragments in the stria-

tum of the PD patient. The first presentation of these studies immediately

provoked the question whether enough basic studies had been performed

to justify such an experimental clinical treatment. Adrenal implantation in

rat models for the parkinsonian dopaminergic failure of the brain have

shown reversal of the motor symptoms, but is it enough evidence to justify

a clinical trial? One of the fundamental requirements in clinical research is

that a sufficient body of animal studies, in particular those carried out in

non-human primates, be reported before trials on human beings can be

performed. It will, however, always be difficult to determine what volume

of animal results justifies the intracranial application of cells or genes in