All of us - kids, mom and dad - went to North Carolina to meet with the team.
We met for dinner on the arival day with Steve and Tom.
Next day we had a tour of the labs and more meeting with the staff.
As the result of meetings and discussions the following work was done and planned:
- AGU skin fibroblasts were received by lab and the work of growing/collecting/preserving for test had began;
- Virus carries is being constracted;
- Gene to be inserted later on, when fibroblasts would be ready for testing;
- Mice will be ordered as soon as checks will be cleared; Mice ordered on week 2, 2013.
- Checks are written for 19 500 US$ on December 19th; additional 19 500US$ will be deposited in April;
- Had discussion with JS in regards to joint work with Franch lab; he is glad to do it. French deligation will be visiting in January and will discuss more detail;
After the order, mice will be received by the lab in ~2 month. It will take another 2-3 month to establish the mice colony, before they can be injected with the virus. Work with fibroblasts will be done before mice are injected.
Started to work with IT professional on new website.
Tuesday, January 15, 2013
Sunday, November 4, 2012
Gene therapy for AGU mice
Here are few articles that I found referring to gene therapy trials in AGU mice. Looks like all were pretty successful.
"We have successfully performed enzyme replacement and gene therapy (with retrovirus-mediated transfer of the AGA gene) both in AGU patients primary fibroblasts and in neural cells of murine origin. Cross-correction between treated and untreated cells could also be achieved, suggesting that only a relatively small number of treated cells would be sufficient to correct the deficient enzyme activity in vivo." AGA, like lysosomal enzymes in general, are good targets for gene therapy since they move from cell to cell using the mannose-6-phosphate receptor. Consequently, only a minority of target cells need to be corrected. Here, we wanted to determine which cell type, neurons or glia would better produce AGA to be transported to adjacent cells for use in possible treatment strategies. Adenoviruses containing tissue-specific glial fibrillary acidic protein (GFAP) promoter and neuron-specific enolase (NSE) promoter were generated to target expression of AGA in Aga-deficient mouse primary glial and neuronal cell cultures. AGA promoter was shown to be a very powerful glia promoter producing 32 times higher specific AGA activity in glia than in neurons. GFAP and NSE promoters also produced a clear overexpression of AGA in glia and neurons, respectively. Interestingly, both the NSE and GFAP promoters were not cell-specific in our system. The amount of exocytosed AGA was significantly higher in glial cells than neurons and glial cells were also found to have a greater capacity to endocytose AGA.
"We have successfully performed enzyme replacement and gene therapy (with retrovirus-mediated transfer of the AGA gene) both in AGU patients primary fibroblasts and in neural cells of murine origin. Cross-correction between treated and untreated cells could also be achieved, suggesting that only a relatively small number of treated cells would be sufficient to correct the deficient enzyme activity in vivo." AGA, like lysosomal enzymes in general, are good targets for gene therapy since they move from cell to cell using the mannose-6-phosphate receptor. Consequently, only a minority of target cells need to be corrected. Here, we wanted to determine which cell type, neurons or glia would better produce AGA to be transported to adjacent cells for use in possible treatment strategies. Adenoviruses containing tissue-specific glial fibrillary acidic protein (GFAP) promoter and neuron-specific enolase (NSE) promoter were generated to target expression of AGA in Aga-deficient mouse primary glial and neuronal cell cultures. AGA promoter was shown to be a very powerful glia promoter producing 32 times higher specific AGA activity in glia than in neurons. GFAP and NSE promoters also produced a clear overexpression of AGA in glia and neurons, respectively. Interestingly, both the NSE and GFAP promoters were not cell-specific in our system. The amount of exocytosed AGA was significantly higher in glial cells than neurons and glial cells were also found to have a greater capacity to endocytose AGA.
Degradation and Turnover of Glycans
For me, not having a biological background, sites that give explanations of the complex and logical biological information for free are always a pot of gold.
Here is a very interesting one that I read today: Degradation and Turnover of Glycans.
It gave me the explanation of glycoprotein degradation sequence, and also have other interesting information.
Here is a very interesting one that I read today: Degradation and Turnover of Glycans.
It gave me the explanation of glycoprotein degradation sequence, and also have other interesting information.
Thursday, October 18, 2012
"It's today, it's today!!! Today is the day!!!"
The phrase is from one of my favorite movies "Stuart Little". And then George asks: " How do you know that you are picking the right one?" and he answers "I guess you'll just know....."
And here we are. Today is the day when we are picking up a University Research Center (URC) to start the research for AGU Gene therapy. There are will be a lot of new things to accomplish and many things to start, unknown and new to us; but with drive and energy to make the other person's life better, we can do it!
And here we are. Today is the day when we are picking up a University Research Center (URC) to start the research for AGU Gene therapy. There are will be a lot of new things to accomplish and many things to start, unknown and new to us; but with drive and energy to make the other person's life better, we can do it!
Wednesday, October 10, 2012
Literature Review #2
Some articles I found interesting and might be applicable to our research (Oct 10, 2012):
1. Gene therapy progress and prospects: gene therapy of lysosomal storage disorders
http://www.nature.com/gt/journal/v10/n16/abs/3302092a.html
The LSDs are monogenic and several small and large, representative animal models of the human diseases are available. Further, the successful reconstitution of only low and unregulated tissue levels of the affected lysosomal enzymes are expected to be sufficient to correct the disease at least in the case of some of the LSDs. For these reasons, they are perceived as good models for the evaluation of different gene delivery vectors and of different strategies for treating chronic genetic diseases by gene transfer. In this review, we will highlight the progress that has been made over the past 2 years in preclinical research for this group of disorders and speculate on future prospects.
2. CNS-directed gene therapy for lysosomal storage diseases
http://onlinelibrary.wiley.com/doi/10.1111/j.1651-2227.2008.00660.x/abstract;jsessionid=BDFC0264422B81ACD25FAB6DD547374C.d04t02?deniedAccessCustomisedMessage=&userIsAuthenticated=false
Gene therapy represents a promising approach for the treatment of CNS disease as it has the potential to provide a permanent source of the deficient enzyme to CNS. Direct intracranial injection of viral gene transfer vectors has resulted in reduced lysosomal storage and functional improvement in certain small (rodent) and large (canine and feline) animal models of LSDs. The addition of protein transduction domains (PTDs) to the recombinant enzymes increased the distribution of enzyme and the extent of correction. Therapeutic levels of lysosomal enzymes can also be delivered to distant sites in the brain by anterograde and retrograde axonal transport. Finally, combining disparate approaches such as BMT and CNS-directed gene therapy can increase treatment efficacy in LSDs with severe CNS disease that are refractory to more conventional approaches
3. Immune Responses to AAV in Clinical Trials (to download)
http://dx.doi.org/10.2174/156652311796150354
4. Potential of AAV vectors in the treatment of metabolic diseaseAAV vectors in the treatment of metabolic disease
http://www.nature.com/gt/journal/v15/n11/full/gt200864a.html
Achieving adequately widespread transduction within the central nervous system, however, remains a major challenge, and will be critical to realization of the therapeutic potential of gene therapy for many of the most clinically troubling metabolic disease phenotypes. Despite the relatively low immunogenicity of AAV vectors, immune responses are also emerging as a factor requiring special attention as efforts accelerate toward human clinical translation. Four metabolic disease phenotypes have reached phase I or I/II trials with one, targeting lipoprotein lipase deficiency, showing exciting early evidence of efficacy.
5. Review: Clinical Trial Outcomes
Gene therapy in neurology: review of ongoing clinical trials
http://www.future-science.com/doi/abs/10.4155/cli.12.47
Gene therapy has entered into its third decade since the first human clinical trial in 1990. The advances in clinical arenas, the successes in this field and the remaining obstacles are highlighted.
6. Gene Therapy Approaches for Lysosomal Storage Disease: Next-Generation Treatment
http://online.liebertpub.com/doi/abs/10.1089/hum.2012.140?mi=3fd470&af=R&prevSearch=allfield%253A%2528integrative+medicine%2529&filter=multiple&nh=20&searchText=integrative+medicine
Since the advent of enzyme replacement therapy (ERT) to manage some LSDs, general clinical outcomes have significantly improved; however, treatment with infused protein is lifelong and continued disease progression is still evident in patients. Viral gene therapy may provide a viable alternative or adjunctive therapy to current management strategies for LSDs. In this review, we discuss the various viral vector systems that have been developed and some of the strategy designs for the treatment of LSDs.
1. Gene therapy progress and prospects: gene therapy of lysosomal storage disorders
http://www.nature.com/gt/journal/v10/n16/abs/3302092a.html
The LSDs are monogenic and several small and large, representative animal models of the human diseases are available. Further, the successful reconstitution of only low and unregulated tissue levels of the affected lysosomal enzymes are expected to be sufficient to correct the disease at least in the case of some of the LSDs. For these reasons, they are perceived as good models for the evaluation of different gene delivery vectors and of different strategies for treating chronic genetic diseases by gene transfer. In this review, we will highlight the progress that has been made over the past 2 years in preclinical research for this group of disorders and speculate on future prospects.
2. CNS-directed gene therapy for lysosomal storage diseases
http://onlinelibrary.wiley.com/doi/10.1111/j.1651-2227.2008.00660.x/abstract;jsessionid=BDFC0264422B81ACD25FAB6DD547374C.d04t02?deniedAccessCustomisedMessage=&userIsAuthenticated=false
Gene therapy represents a promising approach for the treatment of CNS disease as it has the potential to provide a permanent source of the deficient enzyme to CNS. Direct intracranial injection of viral gene transfer vectors has resulted in reduced lysosomal storage and functional improvement in certain small (rodent) and large (canine and feline) animal models of LSDs. The addition of protein transduction domains (PTDs) to the recombinant enzymes increased the distribution of enzyme and the extent of correction. Therapeutic levels of lysosomal enzymes can also be delivered to distant sites in the brain by anterograde and retrograde axonal transport. Finally, combining disparate approaches such as BMT and CNS-directed gene therapy can increase treatment efficacy in LSDs with severe CNS disease that are refractory to more conventional approaches
3. Immune Responses to AAV in Clinical Trials (to download)
http://dx.doi.org/10.2174/156652311796150354
4. Potential of AAV vectors in the treatment of metabolic diseaseAAV vectors in the treatment of metabolic disease
http://www.nature.com/gt/journal/v15/n11/full/gt200864a.html
Achieving adequately widespread transduction within the central nervous system, however, remains a major challenge, and will be critical to realization of the therapeutic potential of gene therapy for many of the most clinically troubling metabolic disease phenotypes. Despite the relatively low immunogenicity of AAV vectors, immune responses are also emerging as a factor requiring special attention as efforts accelerate toward human clinical translation. Four metabolic disease phenotypes have reached phase I or I/II trials with one, targeting lipoprotein lipase deficiency, showing exciting early evidence of efficacy.
5. Review: Clinical Trial Outcomes
Gene therapy in neurology: review of ongoing clinical trials
http://www.future-science.com/doi/abs/10.4155/cli.12.47
Gene therapy has entered into its third decade since the first human clinical trial in 1990. The advances in clinical arenas, the successes in this field and the remaining obstacles are highlighted.
6. Gene Therapy Approaches for Lysosomal Storage Disease: Next-Generation Treatment
http://online.liebertpub.com/doi/abs/10.1089/hum.2012.140?mi=3fd470&af=R&prevSearch=allfield%253A%2528integrative+medicine%2529&filter=multiple&nh=20&searchText=integrative+medicine
Since the advent of enzyme replacement therapy (ERT) to manage some LSDs, general clinical outcomes have significantly improved; however, treatment with infused protein is lifelong and continued disease progression is still evident in patients. Viral gene therapy may provide a viable alternative or adjunctive therapy to current management strategies for LSDs. In this review, we discuss the various viral vector systems that have been developed and some of the strategy designs for the treatment of LSDs.
Saturday, October 6, 2012
Literature Review
Some articles I find interesting and might be applicable to our research:
1. Lysosomal enzyme can bypass the blood–brain barrier and reach the CNS following intranasal administration
http://www.sciencedirect.com/science/article/pii/S1096719212000315
A novel non-invasive route of delivery for lysosomal enzyme to the CNS. Delivery of IDUA to the brain by intranasal administration of laronidase. Delivery of IDUA to the brain by intranasal administration of an AAV vector.
2. Adeno-associated virus (AAV) gene therapy for neurological disease
This review highlights the strategies employed for improving direct and peripheral targeting of therapeutic vectors to CNS tissue, and considers the significance of cellular and tissue transduction specificity, transgene regulation, and other variables that influence achievement of successful therapeutic goals.
3. Strategies for delivery of therapeutics into the central nervous system for treatment of lysosomal storage disorders
http://www.springerlink.com/content/3n8465731114235w/
Approaches to overcome constraints of CNS discussed, among which include modalities of local administration, strategies to enhance the blood-CNS permeability, intranasal delivery, use of exosomes, and those exploiting targeting of transporters and transcytosis pathways in the endothelial lining.
4. The advent of AAV9 expands applications for brain and spinal cord gene delivery
http://informahealthcare.com/doi/abs/10.1517/14712598.2012.681463
Systemic AAV9 gene transfer yields remarkably consistent neuronal expression, though only in early development.
5. Gene Therapy Approaches for Lysosomal Storage Disease: Next-Generation Treatment
http://www.ncbi.nlm.nih.gov/pubmed/22794786
In this review, we discuss the various viral vector systems that have been developed and some of the strategy designs for the treatment of LSDs.
6. Gene therapy and neurodevelopmental disorders
http://www.sciencedirect.com/science/article/pii/S0028390812002821
New developments in AAV vector design are permitting global CNS gene delivery. Expression can be modulated by optimizing both the capsid and genome of vectors. Lysosomal storage diseases represent an immediate target for gene therapy.
7. Delivering drugs to the central nervous system: an overview
http://www.springerlink.com/content/m0jm1117g30487nl/
The goal of this workshop was to present ways to deliver therapeutics to the brain, including the limitations of each method, and describe ways to track their delivery, safety, and efficacy. Solving the problem of delivery will aid translation of therapeutics for patients suffering from neurodegeneration and other disorders of the brain.
8. Adenovirus-Associated Virus Vector–Mediated Gene Transfer in Hemophilia B
http://www.nejm.org/doi/full/10.1056/nejmoa1108046#Background
Peripheral-vein infusion of scAAV2/8-LP1-hFIXco resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype, with few side effects. Although immune-mediated clearance of AAV-transduced hepatocytes remains a concern, this process may be controlled with a short course of glucocorticoids without loss of transgene expression.
Wednesday, October 3, 2012
Gene Therapy thoughts
As I understand the procedure for the gene therapy is as following:
General process for of the most gene therapy studies is:
1. "Normal" gene is inserted into the genome to replace an "abnormal," disease-causing gene.
2. A carrier molecule called a vector must be used to deliver the therapeutic gene to the patient's target cells.
3. Target cells such as the patient's liver or lung cells are infected with the viral vector.
4. New functional protein product from the therapeutic gene restores the target cell to a normal state.
Applying this process to our specific needs:
1. AGA gene is known and very well defined in the literature. There are a lot of data available. I put some more info on the website.
The gene is 11 734 bp long, containing 9 exons. It’s been reported that mutations occur in all 9 exons, but 5th; so we should target at replacing the whole gene to cover all the variations of AGU disease.
Cells (RNA) copy only the information carried by exons to make proteins. So, the length of the DNA material needed to make AGA enzyme is about 255+154+113+113+115+76+108+134+1039=2107 bp. It is a very good news!!!
2. There are different types of viruses used as gene therapy vectors. Mostly commonly the following viruses are used: retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses. The reported drawback of the viruses are:
a. Short-lived nature of gene therapy
b. Immune response
c. Problems with viral vectors
d. Multigene disorders – not an issue in our case, single gene, AGA
For the first three issues, one particular virus can solve them. It is adeno-associated virus (AAV).
AAVs are a class of small, single-stranded DNA viruses that can insert their genetic material at a specific site on chromosome 19. It inserts into genetic material, so will live for a long time in a body and only single injection might be needed. It has no reported immune response problems. You can read more general information about it in English and a little in French (sorry).
We probably will be interested in working with AAV type 2. Serotype 2 (AAV2) has been the most extensively examined so far. AAV2 presents natural tropism towards skeletal muscles, neurons, vascular smooth muscle cells (that exactly what we need!) and Studies have shown that serotype 2 of the virus (AAV-2) apparently kills cancer cells without harming healthy ones. (we like that one, too!)
The reported problem with AAV is the small size, which is about 5000 bp, meaning that the size of gene/molecule that AAV can carry must be less than 5000 bp. It is a small molecule and a lot of genes are much larger.
That is where the size of AGA exons comes. The size of genetic material required to make a good AGA protein/enzyme is only 2107 bp. So, it should fit into AAV!!!!! There are methods to cut a gene to exons and make a smaller gene, something that called plasmid DNA and cDNA. Here is an example of an 11000 bp gene that they were able to put into AAV. There are also companies in USA that make plasmid DNA commercially.
3. Before human trials, mouse and human skin fibroblast trials should be done. We need to consult and figure out the best place to do it from approval/legal point of view.
Also, I found the article that talk about gene therapy for AGU work done in Finland and France in 1998 already. I don’t think they finished the work. The results were very good! You can download the full article through the link. (maybe just wrong vector-carrier, so we’ll fix it)
4. And we wait and see the results!
Let me know your ideas and thought. JL
Possible treatments
There three main types of treatments for LSDs, including AGU
- Enzyme Replacement Therapy (ERT)
- Gene Replacement Therapy (GRT)
- Bone Marrow Transplant (BMT)
We are going (and should) look into all possibilities.
Monday, October 1, 2012
Start
Once upon a time, there lived several happy and dedicated families in different countries around the world.
They did not know each other, but they were somewhat similar. They shared the love, passion and concern for their children. And the kids that these parents have were kind of similar. They were sweet, cute and lovable little kids.
When the kids went to school, more concern took over parents. Although the kids were attending normal school, playing with all other children, they were a little slow.
The parent never gave up in the search for the answer to their concern. And one day they got the news. Children have a rare autosomal recessive Lysosomal Storage Disorder (LSD) called Aspartylglucosaminuria, the diseased that has no cure and not much information available about it.
When the initial shock wore off, the parents got together to find the cure for the kids.
This is the blog of their endeavor and journey.
They did not know each other, but they were somewhat similar. They shared the love, passion and concern for their children. And the kids that these parents have were kind of similar. They were sweet, cute and lovable little kids.
When the kids went to school, more concern took over parents. Although the kids were attending normal school, playing with all other children, they were a little slow.
The parent never gave up in the search for the answer to their concern. And one day they got the news. Children have a rare autosomal recessive Lysosomal Storage Disorder (LSD) called Aspartylglucosaminuria, the diseased that has no cure and not much information available about it.
When the initial shock wore off, the parents got together to find the cure for the kids.
This is the blog of their endeavor and journey.
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