Advances in Gene Therapy for the Treatment of Neurological Disorders

 

                Advances in Gene Therapy  for the Treatment of Neurological Disorders

Abstract

Motivation: Gene Therapy is a latest technology by which diseases are treated by introducing genes. It is especially used to treat neurological disorders. Over the past 20 years more development has occurred in using gene therapy in clinical field to cure neurological disorders. It has become a hopeful for treatment of a number of CNS disorders which were previously considered as incurable. Clinical trials have been performed for the past 20 years to cure diseases such as Lysosomal Storage Diseases, Alzheimer’s disease, Parkinson’s disease, Spinal Muscular Atrophy. Especially to cure Brain Tumors a lot of trials are being done and in process. There are different approaches which are utilized in gene therapy. It involves replacement of gene for a particular inherited disorder or modification of the defective gene in some disorders. By using gene therapy techniques nervous system is modified by different methods which include delivery of specific genes and sequence targeted molecules. Gene Therapy utilizes Recombinant Viral Vectors which serve as vehicles for genes transfer and the most efficient means for gene expression in CNS. These viral vectors include Adeno-Associated Viral Vectors (AAV), Retroviral Vectors, Adeno Viruses and Herpes Simplex Viral Vector (HSV). These Vectors are modified in such a way that they become nontoxic and carry desired genes which are used to cure a disease. These vectors are inserted into the CNS either directly into brain parenchyma tissue or via CSF or may be through intra vascular pathway. Non-viral vectors are also being made now a day. These are DNA or RNA packed in organically modified silica based nanoparticles. Two gene based drugs Gendicine and Glybera are being made so far and are available in the market. More development is being made in the field of gene therapy but there is still need of a breakthrough so that it becomes common and used to cure all neurological diseases.

 

Key Words: Central Nervous System (CNS), Gene Therapy, Blood Brain Barrier (BBB), Alzheimer’s disease (AD), Parkinson’s disease (PD), Epilepsy, Amyotrophic Lateral Sclerosis (ALS), Adeno-associated Viruses (AAV), Retroviruses, Adenoviruses, Herpes Viruses.

 

Results: gene therapy is ideal for curing CNS disorders than using traditional medicines but still more research is needed in this field. A breakthrough is still needed for the treatment become common hence, it is concluded that more work should be done in the field of gene therapy.

 

Introduction

Diseases of Central Nervous System (CNS) are caused by inherited genetic mutations which cause neurodegeneration or impaired functions.  (Michele Simonato, may, 2013).  It is also believed that these neurodegenerative diseases are caused by unfolding of proteins. (Bankiewicz, may, 2013) Environmental causes of CNS diseases are disease related events, injury or inflammation. Traditional medical and surgical procedures don’t give effective treatment.  (Michele Simonato, may, 2013). Entrance of small molecules & proteins from blood into CNS is limited by Blood Brain Barrier (BBB) & skeletal structures enclosing it. BBB has tight junctions between brain microvascular endothelial cells which limit the movement of substances through it. (William J. Bowers1, march, 2011), (Kerstin B. Kaufmann, september,2013), (Boris Kantor*, 2014)Gene Therapy has contributed in this regard to treat these neurological disorders.  (Michele Simonato, may, 2013). The concept of using gene therapy for the treatment of human diseases was given during 1980s. (Sena-Esteves, october, 2010) Before that the idea of Gene Therapy to treat human diseases was given by Friedmann & Roblin in 1972. (Thomas B. Lentz, november, 2012). Neurological Disorders such as Alzheimer’s Disease(AD), Parkinson’s Disease(PD), Epilepsy, Amyotrophic Lateral Sclerosis(ALS) have been studied to be cured using Gene Therapy. (Thomas B. Lentz, november, 2012) Glioma tumors have been a major problem in neurologinal disorders. Attempts have also been made to cure glioma using gene therapy techniques. (William J. Bowers1, march, 2011), (Alex Tobias, february. 2013)Chronic pain which has been a great problem for people has been treated using multiple gene therapy techniques. (William F. Goins, Nov, 2012), (Jean-Marc G Guedon†, may,2015) Sandhoff Disease is autosomal recessive neurogenerative disorder of CNS also treated by gene therapy.  (Hannah E. Rockwell1, 2015).

In gene therapy genes are inserted into target cells by using vectors.  (Boris Kantor* R. M., july, 2015) Viruses are used as vectors to deliver genes to cells of CNS. These viruses are Adeno-associated Viruses (AAV), Retroviruses, Adenoviruses & Herpes Viruses. Viruses are simplest organisms which have properties to be used as vectors for delivering of genes to cells. The structure of a virion carries & protects the genetic information (DNA & RNA) which provides the stability to this genetic material. Viruses have determinants which specify cells in which viruses will transfer specific gene. Viruses can express genes in the infected cells. Viruses can replicate within the host cell so that the desired gene also replicates in the host cell. Ideal vectors are those which do not carry genes of disease but only have desired genes. AAV is a good viral vector as it is easily prepared, do not cause disease and can easily cause transduction in many tissues of CNS. Retroviral vectors are also easily prepared & can target specific cells but there is limitation that they cause mutagenesis. Adenoviruses & herpes viruses are also used but more research is needed to improve them. (Thomas B. Lentz, november, 2012), (Steven J Gray1, october, 2010), (Boris Kantor* R. M., july, 2015), (Boris Kantor*, 2014)

Figure:        Genetic therapy for the nervous system (Hum Mol Genet et.al 2011).

 

Routes of gene delivery to the CNS. CED of viral vectors into the brain improves considerably their distribution in target structures and hence transduction volumes. This technique can yield volumes of transduced cell distribution 3–3.5-fold larger than the infused volume, which is highly significant for human applications. Viral vectors or secreted transgene products (growth factors, lysosomal enzymes) can be further distributed from the primary target structure by axonal transport (top left diagram). Infusion of recombinant proteins or oligonucleotides into the brain ventricular system, or intrathecal space, leads to widespread CNS distribution via CSF flow. An alternative strategy is to use viral vectors to engineer ependymal cells lining the ventricles or choroid plexus cells to secrete therapeutic proteins into CSF (top right diagram). The BBB with its many constituents has thwarted most gene transfer vehicles from entering the brain from the vasculature. In recent years, PILs and a new generation of viral vectors (AAV and SV40) have been shown to mediate efficient CNS gene transfer after i.v. infusion in newborn and adult animals (bottom right diagram).

The vectors are inserted into the brain by direct injection into parenchyma tissue of brain. It is done to target the diseased structure of brain. The vectors can also be entered into CNS via CSF in lateral ventricles or intrathecal spaces. Ideal route of delivery of vectors is through vasculature (Steven et.al 2011).

Figure:  Genetic therapy for the nervous system (Hum Mol Genet et.al 2011).

Schematic representation of existing cellular therapies, including iPS cells, which require vector-based strategies to generate neuroprecursor cells and neurons to ultimately treat neurodegenerative diseases. A variety of cellular therapies have been devised to repair and replace degenerated neuronal networks within the CNS. These strategies utilize multiple sources of neurons and neuroprecursor cells, including fetal brain, pre-implantation embryos, mesenchymal stem cells and iPS cells. The generation of iPS cells requires the use of viral vectors to deliver multiple genes key for the molecular reprogramming of patient fibroblasts. These iPS cells can be differentiated into neurons or neuroprecursor cells that are subsequently transplanted into the diseased brain.

 

Primary metabolic disorders are cured by direct delivery of vectors in brain while secondary metabolic disorders require continuous expression of genes.  (Gao1, July, 2016 ) Non- Viral Vectors are also used in gene therapy. These are Plasmid DNA Nano particles. As naked DNA is unstable, so it is not directly injected into brain parenchyma. Instead it is packed into Nano particles. These are simple, cheap an nontoxic. (Boris Kantor* R. M., july, 2015) Organically modified silica based nanoparticles are used  to pack the desired genes. (William J. Bowers1, march, 2011),  (Rahul Dev Jayant*, october. 2016).

Since the idea of Gene Therapy was given many challenges have been faced by researchers. Continued trials have also made improvements in Gene Therapy procedures.  (Sena-Esteves, october, 2010) It implies many methods to modify the nervous system. These methods include delivery of genes to CNS, Sequence-targeted regulatory molecules & genetically modified cells. (William J. Bowers1, march, 2011)Gene therapy treats a disease by replacing or correcting defective genes. This approach is used in recessive disorders in which defective genes are substituted by correct ones. Some diseases are caused by defective proteins. These are treated by simply suppressing the expression of their genes into proteins. In many diseases of CNS factors are introduced which cause the symptoms of the disease to be delayed or suppressed. (Sena-Esteves, october, 2010)

 

Related Work:

Use of Vectors in curing Neurological Diseases:

AAV vectors are being used in clinical trials to treat Alzheimer’s disease, Parkinson’s Disease, Epilepsy, Amyotrophic Lateral Sclerosis (ALS). Among AAV Vectors Especially serotype 2 (AAV2) is used. Retroviral Vectors are second most commonly used vectors especially in ex vivo transduction of cells to express nerve growth factor (NGF). Lentiviral Vectors are used in clinical trials to treat Parkinson’s disease. (Thomas B. Lentz, november, 2012)

 

Lysosomal Storage Diseases:

These are storage diseases which are caused due deficiency of lysosomal enzymes. These are monogenic diseases. Successful clinical trials have been done on animals for the cure of these diseases.

Two approaches have been used in animal models.

        i.         1. Intraparenchymal infusion of recombinant viral vectors in brain cells.

      ii      2. Successful Bone marrow transplantations in mouse models with ex vivo lentivirus vector modified-autologous (HSCs). These enzymes are wide spread when AAV-mediated modifications are carried out in structures such as cerebella nuclei, ventral tegmental area or thalamus.

Now these are tested on human in clinical trials for the treatment of lysosomal storage diseases. (William J. Bowers1, march, 2011)

Alzheimer’s disease & other Amyloidopathies :

It is a neurodegenerative disorder in which memory loss occurs and there is no cure of it.

Tuszynski & colleagues delivered nerve growth factor (NGF) prototypical neurotrophin in rodents and non-human primates using retrovirus vector-transduced fibroblast grafts. The result was positive and restored cholinergic neurons in these experimental models. Phase I clinical trials were carried out using ex-vivo NGF gene therapy. It was very successful.  (William J. Bowers1, march, 2011)

Ceregene carried out another gene therapy approach in which phase I & II clinical trials were carried out using AAV vectors to produce NGF. (William J. Bowers1, march, 2011)

Spinal Muscular Atrophy (SMA):

It is an autosomal recessive disease caused by loss of function of motor neuron gene SMN1. It leads to degeneration of motors neurons & mortality of infants. Gene Therapy approach used replacement of the missing gene with intravenous administration of AAV9 vector. It delivered SMN1 cDNA to spinal cord in mouse model. It resulted in correction of motor function & survival of mouse. (William J. Bowers1, march, 2011)

Amyotrophic Lateral Sclerosis (ALS):

It is a muscle weakness resulting from loss of motor neurons in brain & spinal cord. It is caused by mutations in gens which produce proteins with toxic functions. When growth factor was introduced in mouse model it increased the survival but did not end the disease. It delayed the symptoms of disease but the disease continued its progression. (William J. Bowers1, march, 2011)

 

Brain Tumors:

Glioma Tumors i.e glioblastoma GBM is the most common adult brain tumor which is untreatable. It is invasive in brain brain tissue. Therapies have been done on experimental models of brain to eliminate tumor mass and inject virus vectors to kill remaining tumor cells. (William J. Bowers1, march, 2011), (Alex Tobias, february. 2013).

Following strategies were used to treat brain tumor.

     i.            Prodrug Activation:

  Viral vectors are introduced which produce enzyme to activate prodrugs into active chemotherapeutic agents in the tumor to cure it.

   ii.            Viral Oncolysis:

In this strategy mutant HSV-1 and adenoviruses are introduced which carry therapeutic genes.

 

 iii.            Cellular Delivery:

In this strategy neural stem cells and mesenchymal cells are used to deliver toxic agents to tumor cells.

 

 iv.            Immunotherapy to Target Tumor Antigens:

It is used to make immune system strong. Antigen EGFRvIII is introduced as vaccination with oncolytic HSV vectors & cyclophosphamide.

 

   v.            Zone of Resistance:

This increases the resistance of brain to tumor. AAV vectors in brain produce interferon-beta which suppress growth of tumor. (William J. Bowers1, march, 2011)

 

Gene Therapy in treatment of Chronic Pain:

Chronic pain have affected about 80 million Americans in their life. It is caused by inflammation or by damage caused to nerve. Gene therapy has been used to treat chronic pain at the level of spinal cord. Phase I human trial was carried out on cancer patients having pain from bone metastasis. Herpes Simplex Viral Vector was used which carried human pre-proenkephalin (hPPE) gene which produced opiod peptides met- & lew-eukephalin (ENK). This showed positive response in relieving pain in patients for up to 1 month after injection. (10) Dr. Tao group showed that restoring kv,2 expression lessen the chronic pain in sensory neurons. (Jean-Marc G Guedon†, may,2015).

 

Figure Nerve injury-induced Kv1.2 downregulation triggered by myeloid zinc finger protein 1 (MZF1)-mediated Kv1.2 antisense (AS) RNA expression in the injured dorsal root ganglion (DRG). (A) Under normal conditions, Kv1.2 mRNA that is transcribed from the genome is translated into Kv1.2 protein, resulting in normal expression of Kv1.2 channel at DRG neuronal membrane. (B) Under neuropathic pain conditions, peripheral nerve injury promotes the expression of the transcription factor MZF1 in DRG. The increased MZF1 binds to the promoter region of Kv1.2 AS RNA gene and triggers its expression. The latter specifically and selectively inhibits the expression of Kv1.2 mRNA via extensive overlap of their complementary regions, leading to a reduction in the membrane expression of Kv1.2 only, not other Kv subunits (e.g., Kv1.1), in the DRG neurons.

Adeno-associated virus (AAV) mediated transfer of Kv1.2 sense RNA for the reduction of DRG neuronal excitability. (A) Before AAV injection into the DRG of rats with peripheral nerve injury, a nerve injury-induced increase in DRG Kv1.2 AS RNA triggered by MZF1 knocks down expression of Kv1.2 mRNA and protein, resulting in an increase in DRG neuronal excitability under neuropathic pain conditions. (B) After AAV injection into the DRG of rats with peripheral nerve injury, AAV mediated transfer of full length Kv1.2 sense (SE) RNA rescues nerve injury-induced DRG Kv1.2 downregulation at the DRG neuronal membrane through not only its direct translation into Kv1.2 protein but also its indirect blockage of nerve injury-induced increase in Kv1.2 AS RNA expression via extensive overlap of their complementary regions. AAV mediated transfer of Kv1.2 SE RNA fragment (-311 to +40) also rescues nerve injury-induced DRG Kv1.2 downregulation through its blockage of nerve injury-induced increase in Kv1.2 AS RNA expression via partial overlap of their complementary regions, although this RNA fragment cannot be translated into Kv1.2 protein. Maintaining normal Kv1.2 expression at DRG neuronal membrane reduces nerve injury-induced neuronal hyperexcitability at DRG neurons and consequently decreases spinal central sensitization, resulting in neuropathic relief.

 

Rett Syndrome:

In Rett Syndrome, slowing of development occurs such as slow brain & head growthloss of hand movements & use of hands. Problems with walking & intellectual disability also occur. Rett syndrome has been treated with gene therapy.MeCP2 gene is inserted in neurons & glial cells which resulted in lessening the disease symptoms.  (Boris Kantor* R. M., july, 2015)

Sandhoff Disease:

It is caused due to mutation of gene which causes accumulation of GM2 in CNS. AAV mediated gene therapy is used to treat this disease. Successful clinical trials have been done on in murine and cat brain models. It showed improvements and hopeful for treating the disease in humans.  (Hannah E. Rockwell1, 2015)

 

Discussion:

Gene Therapy is the most advance treatment strategy used to treat many neurological disorders in clinical field. Two decades back it was impossible to treat neurological disorders. They were considered as inherited and genetic disorders. It was impossible to cure genetic disorders. But in the advancement of genetics it has become possible to even cure there genetic disorders. Researchers first made clinical trials on animal models. Within these 20 years gene therapy has become practically possible. Now many neurological disorders are being treated by using gene therapy. Different strategies were implied such as to replace a defected gene or to introduce new gene into the CNS. Researchers faced many problems in the development of vectors. But their continued efforts made possible to make a variety of vectors to be used as gene carriers to the brain. Now a days work is being done on producing  non-viral vectors. Routes of administration were also considered a problem in the past but now there are many routes by which genes are inserted in CNS. In other words, many efforts were made by researchers to develop better gene therapy techniques.

Future Perspectives:

A lot of scope is remaining in this field. There is a need of a lot of work in the area of gene therapy. More research should be done to make the process more better and safe. Especially in the field of producing non-viral vectors. A lot of effot is required in this field to produce efficient an safe vectors. More drugs should be made so that the treatment becomes common. There is still need for more research in the field of making genetic drugs for common people.

 

Conclusion:

In Short, many clinical trials have been made in the field of gene therapy to cure CNS disorders for the past 20 years. A lot of improvement has also been made in this field but there is still need of more improvement in this regard.

The idea of using gene therapy is ideal for curing CNS disorders than using traditional medicines but still more research is needed in this field. A breakthrough is still needed for the treatment become common Hence, it is concluded that more work should be done in the field of gene therapy

 

References

1.      Bowers, W. J., Breakefield, X. O., & Sena-Esteves, M. (2011). Genetic therapy for the nervous system. Human molecular genetics, 20(R1), R28-R41.

2.      Breakefield, X. O., & Sena-Esteves, M. (2010). Healing genes in the nervous system. Neuron, 68(2), 178-181.

3.      Gessler, D. J., & Gao, G. (2016). Gene therapy for the treatment of neurological disorders: metabolic disorders. Gene Therapy for Neurological Disorders: Methods and Protocols, 429-465.

4.      Goins, W. F., Cohen, J. B., & Glorioso, J. C. (2012). Gene therapy for the treatment of chronic peripheral nervous system pain. Neurobiology of disease, 48(2), 255-270.

5.      Gray, S. J., Woodard, K. T., & Samulski, R. J. (2010). Viral vectors and delivery strategies for CNS gene therapy. Therapeutic delivery, 1(4), 517-534.

6.      Guedon, J. M. G., Wu, S., Zheng, X., Churchill, C. C., Glorioso, J. C., Liu, C. H., ... & Kinchington, P. R. (2015). Current gene therapy using viral vectors for chronic pain. Molecular pain, 11(1), 27.

7.      Jayant, R. D., Sosa, D., Kaushik, A., Atluri, V., Vashist, A., Tomitaka, A., & Nair, M. (2016). Current status of non-viral gene therapy for CNS disorders. Expert opinion on drug delivery, 13(10), 1433-1445.

8.      Kantor, B., Bailey, R. M., Wimberly, K., Kalburgi, S. N., & Gray, S. J. (2014). Methods for gene transfer to the central nervous system. Advances in genetics, 87, 125.

9.      Kantor, B., McCown, T., Leone, P., & Gray, S. J. (2014). Clinical applications involving CNS gene transfer. Advances in genetics, 87, 71.

10.  Kaufmann, K. B., Büning, H., Galy, A., Schambach, A., & Grez, M. (2013). Gene therapy on the move. EMBO molecular medicine, 5(11), 1642-1661. (Kerstin B. Kaufmann, september,2013)

11.  Lentz, T. B., Gray, S. J., & Samulski, R. J. (2012). Viral vectors for gene delivery to the central nervous system. Neurobiology of disease, 48(2), 179-188.

12.  Rockwell, H. E., McCurdy, V. J., Eaton, S. C., Wilson, D. U., Johnson, A. K., Randle, A. N., ... & Cox, N. R. (2015). AAV-mediated gene delivery in a feline model of Sandhoff disease corrects lysosomal storage in the central nervous system. ASN neuro, 7(2), 1759091415569908.

13.  San Sebastian, W., Samaranch, L., Kells, A. P., Forsayeth, J., & Bankiewicz, K. S. (2013). Gene therapy for misfolding protein diseases of the central nervous system. Neurotherapeutics, 10(3), 498-510.

14.  Simonato, M., Bennett, J., Boulis, N. M., Castro, M. G., Fink, D. J., Goins, W. F., ... & Wolfe, J. H. (2013). Progress in gene therapy for neurological disorders. Nature Reviews Neurology, 9(5), 277-291.

15.  Tobias, A., Ahmed, A., Moon, K. S., & Lesniak, M. S. (2012). The art of gene therapy for glioma: a review of the challenging road to the bedside. J Neurol Neurosurg Psychiatry, jnnp-2012.

 

 

 About Author:

Mr. Imran Zafar has completed his Bachelor of Science (BS) degree in Bioinformatics from COMSATS Institute of Information Technology Islamabad Sahiwal campus under supervision of Dr. Ahmad Ali and Master of Science (MS) in Bioinformatics from Department of Bioinformatics and Computational Biology, Virtual University of Pakistan, Lahore, Punjab, Pakistan under supervision of Dr. Muhammad Tariq Pervez. For research work during BS and MS he has also done internships from School of biological Science (SBS), University of Veterinary and Animal Sciences (UVAS) and Center of Excellence in molecular biology (CEMB) Lahore. He has published several research articles and book computers in reputed journals recognized from Higher Education Commission (HEC) of Pakistan.  His research is mainly focused on the field of Bioinformatics, Genomics, Computational Biology and Molecular Biology in the domain of life science to performed computational analysis. He is now working in Ministry of Education as a Science subject instructor in the Department of Education Punjab, Pakistan.