**2.2 Strategies of growth factor delivery**

### **2.2.1 Methods of delivery**

Currently, three different methods of have been used to deliver the growth factor into the motor nervous system to ALS patients or rodent models. The first is subcutaneous injection of the growth factor protein. The obvious advantage of this method is the ease and simplicity to administrate. Some growth factors are pharmaceutically available to treat other neurodevelopmental diseases, such as IGF-1 to treat IGF deficiency in children. This is the reason why it is the only method of delivery that has been tested on human ALS patients. However, a statistically significant result has not been observed in this method of delivery. The only successful case is the North American study on IGF-1 in 1997 (Lai et al., 1997), but was immediately challenged by an almost identical study in Europe in 1998 (Borasio et al., 1998) and other later studies. The failure of this classical method of delivery to alleviate ALS symptoms includes (i) inability of some of the chemical of interest to pass the blood-brain barrier; (ii) unwanted side effects in non-targeted sites, and (iii) a relative short half-life of the protein. The significance of these issues is amplified in the human nervous system because of greater cross-sectional area when compared to rodents. Further penetration is needed for the injected growth factor to reach the deep structure in the brain or spinal cord to give its desired effect. Similar issues are found in clinical trials for patients with Parkinson's disease using the same strategy to deliver growth factors.

The second method is to deliver the chemical of interest by implanting a catheter directly into the site of the brain that needs the growth factor, as seen in a couple Parkinson's disease studies (Gill et al., 2003; Slevin et al., 2005). It is better than the previous method as it overcomes the distance problem seen in large animals. However, there are a couple of drawbacks if this is applied to ALS patients to deliver the growth factor into the spinal cord instead of the brain for Parkinson's disease. The implanted catheter might interrupt the ascending and/or descending white matter track, and the natural movement of the spinal cord in patients increase the shearing forces may cause further damage. Therefore catheter delivery would not be a desirable method of ALS growth factor delivery.

The last approach uses viral vectors to circumvent all those issues. Those viruses include lentivirus (Cisterni et al., 2000; Hottinger et al., 2000; Azzouz et al., 2004), adenovirus (Acsadi et al., 2002; Hasse et al., 2007), and adeno-associated virus (AAV) (Kasper et al., 2003; Wang et al., 2002). They are used because of the ability to deliver genes to non-dividing cells, which includes mature neurons. Thus they are ready to be engineered to encode the therapeutic protein. Extensive studies of AAV delivery of potential drugs to specific brain regions have been published, suggesting viral vector delivery is a practical method.

### **2.2.2 Sites of delivery**

438 Amyotrophic Lateral Sclerosis

G93A and other SOD1 transgenic rodent models as SOD1 mutation is only found in a small proportion of human ALS patients. However, it is still an excellent tool for ALS researchers as transgenic mice have proven to be one of the most useful tools to understand the complexity of neurodegenerative diseases because of their usefulness to unveil underlying mechanisms of the disease and evaluating potential treatments (Rothstein, 2004). In this review we will overview the extensive use of SOD1 transgenic rodent models in ALS

Topics covered in this chapter include growth factor therapy and stem cell therapy for ALS. For growth factor therapy, we will introduce different delivery methods and injection sites. As for stem cell transplantation therapy, we will look into strategies that aim to replace or protect motor neurons. After that, we will summarize studies that utilize stem cells as a tool to deliver growth factors. We will conclude the chapter by looking forward to future

Growth factors are a class of naturally occurring proteins that are capable of stimulating cell growth, proliferation, and differentiation. In development of the nervous system, they are crucial because they are essential for neuronal survival and differentiation. For adults, they are also required in some cases to maintain normal function of the nervous system, but only at very low levels. However, the presence of low levels of growth factors in adult tissues is critical because motor neurons rely on them for survival and repair upon stress and injury. Experiments have been performed to investigate the effect of growth factors on alleviating the symptoms of ALS. Those growth factors includes glial cell line-derived neurotrophic factor (GDNF), insulin growth factor 1 (IGF-1), vascular endothelial growth factor (VEGF), and brain derived neurotrophic factor (BDNF). For each of the growth factors listed above, there are studies on hSOD1-G93A transgenic rodent models that show some degree of improvement, which includes some or all of the following: delay onset, slow disease progression, decrease

Currently, three different methods of have been used to deliver the growth factor into the motor nervous system to ALS patients or rodent models. The first is subcutaneous injection of the growth factor protein. The obvious advantage of this method is the ease and simplicity to administrate. Some growth factors are pharmaceutically available to treat other neurodevelopmental diseases, such as IGF-1 to treat IGF deficiency in children. This is the reason why it is the only method of delivery that has been tested on human ALS patients. However, a statistically significant result has not been observed in this method of delivery. The only successful case is the North American study on IGF-1 in 1997 (Lai et al., 1997), but was immediately challenged by an almost identical study in Europe in 1998 (Borasio et al., 1998) and other later studies. The failure of this classical method of delivery to alleviate ALS symptoms includes (i) inability of some of the chemical of interest to pass the blood-brain

motor neuron loss, preserve neuromuscular junction and prolong survival.

research and how those findings can be transferred to treat human ALS patients.

**1.2 Chapter overview** 

development in the field.

**2. Growth factors and gene therapy in ALS** 

**2.1 Growth factors and the nervous system** 

**2.2 Strategies of growth factor delivery** 

**2.2.1 Methods of delivery** 

Studies have been done to inject vectors encoding the growth factor of interest into two distinctive types of tissues: (i) limb/respiratory muscles and (ii) the connecting motor neurons. In most ALS studies the vectors are injected in the muscle. Although positive results are shown in studies with GDNF and IGF-1, researchers believes that motor neurons may detach from the muscle at early stages of the disease (Fischer et al., 2004), or the cellular transport mechanism is heavily impaired (Williamson & Cleveland, 1999; De Vos et al., 2007). Again due to their large cross-sectional area, retrograde transport is more severely affected in larger mammals when compared to mice, and thus requires a longer distance of transport. This factor may slow the translation of this successful strategy to clinical trials.

To overcome the potential problems of retrograde transport that may be encountered in muscle injections in humans, studies that inject vectors directly to motor neurons within spinal cord has been performed. Surprisingly, only a few studies have been published on this approach and the effect is less significant than the muscle injection studies. In a GDNF study on ALS mice, neuroprotection is only seen on facial but not lumbar motor neurons (Guillot, 2004). Another study supports the above idea by showing that GDNF is neuroprotective when it is overexpressed in skeletal muscles, but has no effect when the growth factor is overexpressed in motor neurons (Li et al., 2007). Disease progression is only slowed when GDNF is expressed in skeletal muscles, but not when it is expressed in the motor neurons.

### **2.3 Insights from growth factor studies to understand ALS disease progression**

Although the ultimate goal of growth factor therapy for ALS is to alleviate symptoms, prolong survival, delay onset, and slow disease progression, during the course of

Stem Cell Application for Amyotrophic Lateral Sclerosis: Growth Factor Delivery and Cell Therapy 441

(Dimos, 2008). However, introduction of new genes during the production of iPS cells may

Mouse ES-derived motor neurons reportedly grow around the ventral horn when transplanted into the spinal cord of rats with impaired motor neurons (Harper et al., 2004). In combination with chemicals that overcome myelin-mediated repulsion and GDNF that stimulates axon guidance towards skeletal muscles, further improvement in survival and engraftment of the transplanted cells was observed. Improvement in motor function of the

Despite the excitement that these transplantation studies brings to the field, the fact that these studies were performed on static models of motor neuron loss does not guarantee success in progressive motor neuron diseases like ALS. In addition, in order for the motor neuron replacement strategy to be successful, the transplanted motor neuron will first need to receive synaptic input from the presynaptic neurons and extend it's axon all the way to the targeted muscle at a rate of 1-3 mm/day, which takes months to years in humans, before innervation to the targeted muscle can be possible (Papadeas & Maragakis, 2009). Therefore

Previously, little attention has been paid to the function of glial cells in the nervous system. However, we now know that glial cells modulate neuronal functions such as glutamate uptake, synaptic plasticity, trophic factor support, and even neuronal transmission (Kirchhoff et al., 2001). Studies also show that motor neuron death in ALS is non-cell autonomous, or mediated by astrocytes and microglia (Hall et al., 1998; Barbeito et al., 2004). Researchers also hypothesize that astrocytes and/or microglia form a positive feedback loop with motor neurons that leads to further propagation of the disease (Rao & Weiss, 2004). Moreover, chimeric mice with increased proportion of healthy, wild type glial cells increase survival of nearby human SOD1 mutant neurons *in vivo* (Clement et al., 2003). Using a CRE-lox system, selective reduction of the mutant gene in microglia and astrocytes in SOD1 transgenic mice slows disease progression,

Additional evidence is provided by stem cell–derived motor neurons/astrocytes co-culture. A study in 2007 shows that primary and ES cell-derived motor neurons are complementary in an *in vitro* motor neuron/astrocytes study for ALS (Nagai et al., 2007). From then on, studies using the following combinations have been performed: hES cell derived motor neurons with primary hSOD1-G93A or wild type mouse primary astrocytes (Di Giorgio et al. 2008); hSOD1- G93A mouse ES derived motor neuron with hSOD1-G93A derived mouse primary astrocytes (Di Giorgio 2007); and hES cells derived motor neuron with primary human astrocytes transfected with hSOD1-G47R genes (Marchetto, 2008). The Marchetto paper also uses that approach to verify a potential drug that has been beneficial in ALS rodent models. The success in this approach provides an easily accessible *in vitro* testing platform for cell-cell interactions in ALS and underlying disease mechanisms. Drug discovery will also accelerate as high

Based on non-cell autonomous nature of motor neuron death in ALS, astrocyte replacement is another feasible strategy for ALS stem cell therapy. Researchers transplant

give rise to additional technical concerns when translating to clinical studies.

motor neuron replacement may not be a legitimate treatment at this moment.

**3.2.1 Non-cell autonomous nature of motor neuron death in ALS** 

but has no effect on disease onset (Boillée et al., 2006b; Yamanaka et al., 2008).

throughput drug screening can be performed on the cultures.

**3.2.2 Astrocyte replacement** 

paralyzed rats was also observed (Despande et al., 2006).

**3.2 The neuroprotection strategy** 

investigation several interesting findings have been observed and may provide insights to better understand the underlying mechanism of the disease. For example, finding growth factors' targets may help us find how the disease is initiated. Currently, the growth factors' targets are not fully known. It could be the degenerating motor neuron itself, the neighboring neuron, or surrounding glial cells. But a recent report about wild type nonneuronal cells extending survival of SOD1 mutant motor neurons in chimeric ALS mice (Clement et al., 2003) may provide adequate evidence showing that the growth factor's target is the supporting glia instead of neurons.

Another point of interest is the similarity of the growth factors that have been used. All GDNF, IGF-1, VEGF, and BDNF interact with receptor tyrosine kinases to produce downstream effects. Experiments have shown that those growth factors indeed work in a similar pathway and mechanism as there is no additional improvement observed when they work in combination (IGF-1 and VEGF) as compared to working individually (Dodge et al. 2010). Another article reports that VEGF promotes motor neuron survival by blocking Caspase through Phosphoinositide 3-kinase/ protein kinase B (PI3K/Akt) pathway (Lunn et al., 2009). Further investigation on the PI3K/Akt pathway may provide clues on how motor neuron death is triggered in ALS.
