Minecraft: Attack of the B Team, Ep. 51 Gene therapy!
In this episode of Attack of the B Team we put our advanced genetics knowledge to use, not good use, just use.... Really the best use for dna? https://www.fa...
By: Dr. Trotter
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Minecraft: Attack of the B Team, Ep. 51 Gene therapy! - Video
Methylmalonic Acidemia (MMA) Gene Therapy - Charles Venditti and Randy Chandler
Dr. Charles Venditti and Dr. Randy Chandler discuss recent developments in gene therapy for methylmalonic acidemia (MMA) - a group of inherited disorders in which the body is unable to process...
By: GenomeTV
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Methylmalonic Acidemia (MMA) Gene Therapy - Charles Venditti and Randy Chandler - Video
Washington, D.C.-basedgene therapy outfit Regenxbio just raised $30 million to bringits platforminto the clinic.
The dollars will help Regenxbio generate clinical proof of concept data, as well as work toward in-licensing new programs. The companys also using the money to beef up its clinical and manufacturing processes, it said in a statement.
Regenxbio has developed what it calls NAV Technology a form of adeno-associated viral gene therapy that treats lysosomal storage disorders and ocular disease. Its got drugs in the pipeline that treat Hurler syndrome, Hunter syndrome, wet age-related macular degeneration and X-linked retinitis pigmentosa.
Heres how it works: In Hurler syndrome, for instance, children dont carry a gene that develops an enzyme called IDUA that breaks down complex sugars. These build up, and ultimately impairmental development, organ function, physical abilities and appearance. The NAV platform delivers a normal copy of the IDUA-producing gene which ultimately embeds itself into a patients DNA in a one-time doze so that patients can produce the enzyme. The research comes out of the University of Pennsylvania, and has been successful in vivo so its a matter of testing its efficacy in a real patient pool.
The companys also out-licensing this technology for other indications, with companies like Baxter, Dimension Therapeutics and Lysogene developing viral vector-based gene therapy for a number of other indications.
The Series C funding was led by Venrock and Brookside Capital, with other investors like Deerfield Management, FoxKiser and Fidelity Biosciences.
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Regenxbio nets $30M to bring its gene therapy to clinical trial
PHILADELPHIA, Penn., January 20, 2015- Spark Therapeutics, a late-stage gene therapy company developing treatments for debilitating genetic diseases, announced today it has initiated a Phase 1/2 clinical trial for the potential treatment of patients with choroideremia (CHM) utilizing its gene therapy product SPK-CHM. The Phase 1/2 trial is an open-label, dose-escalating trial designed to assess the safety and preliminary efficacy of sub-retinal administration of SPK-CHM. The Phase 1/2 trial will be conducted at The Children's Hospital of Philadelphia (CHOP) and the University of Pennsylvania, and has plans to enroll up to 10 patients afflicted with the CHM genetic mutation.
"Spark's groundbreaking announcement today brings real hope of a treatment for blindness caused by choroideremia, and promises to pave the way for treatments of other retinal diseases impacting millions of people around the World," said Dr. Chris Moen, President of the Choroideremia Research Foundation (curechm.org), the leading advocacy and fundraising organization focused on finding a cure for CHM. "The Choroideremia Research Foundation is proud to have provided key pre-clinical funding to Jean Bennett, MD, PhD and her team atthe Perelman School of Medicine at the University of Pennsylvania,that has helped bring us to the gene therapy human clinical trials being announced today."
Dr. Jean Bennett's pre-clinical work, which was funded in part by consistent financial support from the Choroideremia Research Foundation (curechm.org), demonstrated the ability of the SPK-CHM gene therapy to restore REP-1 protein production, membrane trafficking and retinal structure.
"Throughout my career's work developing genetic therapies for inherited retinal dystrophies I have had my target set on a number of different conditions, in particular, choroideremia," said Dr. Bennett, who is also one of Spark's scientific co-founders and a scientific advisor on the SPK-RPE65 clinical trials being conducted at CHOP. "The SPK-CHM program, for the first time, creates the potential for patients to use their vision for longer and see more things."
In addition to evaluating safety, the trial will help define the dose required to achieve stable or improved visual function and identify appropriate endpoints for subsequent clinical trials. With SPK-CHM, Spark is leveraging the experience and technology utilized in the development of its gene therapy for Leber's Congenital Amaurosis (LCA), SPK-RPE65, including the same vector, target cells and route of administration, as well as the same manufacturing process. SPK-RPE65 is currently in a fully-enrolled pivotal Phase 3 clinical trial.
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U.S. Gene Therapy Clinical Trial to treat Choroideremia initiated
Gene Therapy: Hope For The Blind?
In medicine, doctors often talk about treatments, but rarely about cures. Now, gene therapy is offering the potential to actually cure certain diseases and an experimental breakthrough could...
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Gene therapy is a promising strategy to correct hereditary disorders. The approach takes advantage of viral vectors to deliver a corrected version of the mutated gene. Adeno-associated virus (AAV) has many features that make it a favorable vector for gene therapy. In animal models, AAV-mediated gene delivery is generally regarded as safe and has demonstrated efficacy for some genetic diseases. However, a recent study reported an increase in liver cancer in mice after AAV gene therapy. A new publication in the Journal of Clinical Investigation reveals that AAV vector design influences the likelihood of developing cancer in the liver. Charles Venditti and colleagues at the National Institutes of Health looked for the development of hepatocellular carcinoma (HCC) in a large number of mice that had received AAV gene therapy. HCC was associated with the AAV vector integrating within a specific site in the genome and inducing expression of microRNAs and a retrotranposon. Moreover, AAV dose, the choice of enhancer/promoter, and timing of delivery all influenced the HCC incidence. The results of this study provide insight into features that should be considered when designing AAV vectors for gene therapy.
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TITLE:
Vector design influences hepatic genotoxicity after adeno-associated virus gene therapy
AUTHOR CONTACT:
Charles Venditti National Institutes of Health, Bethesda, MD, USA Phone: 301-496-6213; Fax: 301-451-3853; E-mail: venditti@mail.nih.gov
View this article at: http://www.jci.org/articles/view/79213?key=0ff2890aa2b1c1b59bca
Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system.
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Gene therapy-associated cancer incidence depends on vector design
Pre-clinical studies in mice reveal ways to reduce cancer risk with modified treatment
National Institutes of Health researchers have uncovered a key factor in understanding the elevated cancer risk associated with gene therapy. They conducted research on mice with a rare disease similar to one in humans, hoping their findings may eventually help improve gene therapy for humans. Researchers at the National Human Genome Research Institute (NHGRI), part of NIH, published their research in the Jan. 20, 2015, online issue of the Journal of Clinical Investigation.
"Effective and safe gene therapies have the potential to dramatically reverse diseases that are life-threatening for affected children," said NHGRI Scientific Director Dan Kastner, M.D., Ph.D. "This study is an important step in developing gene therapies that can be safely used to benefit patients."
Toxic side effects actually are rarely observed by researchers who have designed gene therapies using an adeno-associated virus (AAV) as a vector to deliver the corrected gene to a specific point in the cell's DNA. AAVs are small viruses that infect humans but do not cause disease. A vector is a DNA molecule of AAV used as a vehicle to carry corrected genetic material into a cell. AAV viruses are uniquely suited for gene therapy applications.
But one prior study did find an association between AAV and the occurrence of liver cancer. The present research addresses this problem in gene therapy for an inherited disease in children called methylmalonic acidemia, or MMA.
For 10 years, NHGRI researchers have worked toward a gene therapy to treat MMA. The condition affects as many as 1 in 67,000 children born in the United States. Affected children are unable to properly metabolize certain amino acids consumed in their diet, which can damage a number of organs and lead to kidney failure. MMA patients also suffer from severe metabolic instability, failure to thrive, intellectual and physical disabilities, pancreatitis, anemia, seizures, vision loss and strokes. The most common therapy is a restrictive diet, but doctors must resort to dialysis or kidney or liver transplants when the disease progresses.
In prior MMA gene therapy studies, researchers showed that mice bred to develop the condition could be restored to health by AAV gene therapy injection shortly after birth. The mice in the study survived into adulthood and were free from the effects of MMA.
"The corrected gene delivered by AAV is the most effective therapy we have developed so far to treat MMA," said Charles Venditti, M.D., Ph.D., senior author and investigator in NHGRI's Genetic and Molecular Biology Branch. "However, we have identified an important safety parameter related to the AAV gene therapy in our mouse models that is critical to understand before we move to human patient trials."
Now, in a long-term follow-up of the treated mice -- after mice reached about two years of age -- the researchers documented a 50-70 percent higher occurrence of liver cancer in AAV-treated mice compared with a 10 percent liver cancer rate in untreated mice. Dr. Venditti's team determined that the AAV vector triggered the cancer.
The research team performed additional experiments to detect where in the mouse genome the AAV vector delivered the corrected gene and how that related to any cancer development. In many mice that developed liver cancer, the AAV vector targeted a region of the mouse genome called Rian, near a gene called Mir341 that codes for a microRNA molecule. MicroRNAs are small, non-coding RNA molecules involved in the regulation of gene expression. When the AAV was inserted near Mir341, the vector caused elevated expression of the gene, which the researchers believe contributed to the occurrence of liver cancer in the mice. The authors note that Mir341 is found in the mouse genome, however, it is not present in humans.
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Spark Therapeutics, which is developing gene therapy treatments for retinal dystrophies and hematologic disorders, announced terms for its IPO on Tuesday. The Philadelphia, PA-based company plans to raise $88 million by offering 5.5 million shares at a price range of $15 to $17. At the midpoint of the proposed range, Spark Therapeutics would command a fully diluted market value of $378 million.
Spark Therapeutics, which was founded in 2013, plans to list on the NASDAQ under the symbol ONCE. J.P. Morgan and Credit Suisse are the joint bookrunners on the deal. It is expected to price during the week of January 26, 2015.
Investment Disclosure: The information and opinions expressed herein were prepared by Renaissance Capital's research analysts and do not constitute an offer to buy or sell any security. Renaissance Capital, the Renaissance IPO ETF (symbol: IPO) or the Global IPO Fund (symbol: IPOSX) , may have investments in securities of companies mentioned.
The views and opinions expressed herein are the views and opinions of the author and do not necessarily reflect those of The NASDAQ OMX Group, Inc.
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Gene therapy biotech Spark Therapeutics sets terms for $88 million IPO
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Newswise PHILADELPHIA The University of Pennsylvania has announced an expanded relationship with Spark Therapeutics, a late-stage gene therapy company developing treatments for debilitating, genetic diseases. As part of the expanded relationship, which included both an exclusive license agreement to certain Penn-owned intellectual property rights and a clinical trial agreement, Penn will be one of the clinical sites for a clinical trial to evaluate the treatment of a rare genetic form of progressive blindness.
This expanded strategic relationship between the two organizations is representative of Penns strong commitment towards direct engagement with the private sector to advance promising technologies, said John Swartley, Associate Vice Provost for Research and Executive Director, Penn Center for Innovation (PCI). When Penn researchers on the cutting edge of their fields are able to partner effectively with innovators in the private sector it has the potential to accelerate the advancement of exciting new approaches for the treatment of disease. PCI serves as the University of Pennsylvanias commercialization center and actively works with the private sector to foster research and development collaborations leveraging Penn technology and research.
Spark today announced the start of Phase 1/2 clinical trial for patients with choroideremia (CHM), which will take place at The Childrens Hospital of Philadelphia and the University of Pennsylvania. CHM is a rare, genetic eye disorder that causes progressive vision loss, ultimately leading to complete blindness. CHM is characterized by deletions or mutations in the CHM gene. It is a degenerative eye disease which affects males that manifests in childhood as night blindness and a reduction in visual field, followed by progressive constriction of visual fields leading, ultimately, to blindness. There is currently no approved drug treatment for the disease.
The launch of this clinical trial is the latest facet of the ongoing partnership between Spark and Penn.Expanding upon an earlier collaboration around SPK-RPE65, in December of 2014, Spark and Penn, through PCI, entered into an exclusive license agreement to certain Penn-owned intellectual property rights, including assets related to the choroideremia program. As a part of the license agreement, Penn received equity shares in Spark and may receive additional milestone payments and royalties on net sales dependent on the success of the SPK-CHM program.
The new trial is designed to assess the safety and preliminary efficacy of sub-retinal administration of SPK-CHM. The investigators plan to enroll up to 10 patients afflicted with the CHM genetic mutation. In addition to evaluating safety, the trial will help define the dose required to achieve stable or improved visual function and identify appropriate endpoints for subsequent clinical trials. The trial will build on the work of the clinical trial teams that have conducted trials of Sparks therapy known as SPK-RPE65, which has been observed in clinical trials to improve vision in patients with rare blinding conditions due to mutations in the RPE65 gene.
I have a particular interest in choroideremia, says Jean Bennett, MD, PhD, the F.M. Kirby Professor of Ophthalmology and director of the Center for Retinal and Ocular Therapy (CAROT) at the Perelman School of Medicine at the University of Pennsylvania, and one of Sparks scientific co-founders. I am thrilled to now be able to test our gene therapy treatments with the potential to help the many men living with this disorder.
"Editors note: The University of Pennsylvania has licensed technology involved in this research to Spark. Dr. Bennett is an inventor of this technology, and may benefit financially."
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Gene Therapy advance online publication 15January2015; doi: 10.1038/gt.2014.124
First-generation (E1 deleted) adenovirus vectors (FG AdVs), which lack the E1 and E3 regions, are popularly used in basic studies to elucidate gene functions, and have been employed for gene therapy.1, 2, 3, 4 Because the DNA fragments of up to about 7 kilobases (kb) in total can be inserted into the AdV genome, the AdVs are frequently used to produce two proteins simultaneously from two independent transgenes expressing both the target gene and the reporter gene, for example. In the studies using the cultured cells and in the animal experiments, the GFP and luciferase are used as the reporters. Recently, positron emission tomography has clinically been used in patients for diagnoses and in experimental animal models. Therefore, the AdVs containing both the therapeutic gene and the positron emission tomography reporter gene would be valuable in the gene therapy fields, because the therapeutic effects, the vector duration and distribution can simultaneously be monitored.5, 6, 7, 8 Probably one would wish for high-titer AdVs with the highest expression for the therapeutic gene and with the second highest for the reporter gene not causing any trouble, if the insertion sites and orientations in the AdV genome can be chosen. However, the titers and the expression levels of the AdVs may considerably be influenced by the sites and orientations of the transgenes. Such information may be very valuable for construction of the best vector, especially in the vector containing both the therapeutic gene and the reporter gene.
The simultaneous expression of two genes could be achieved by inserting the two genes into the E1 site under the control of a single prompter using the internal ribosomal entry sites or using porcine teschovirus-1 2A.9, 10 In the former approach, the expression of the second gene might be influenced by the sequences between internal ribosomal entry sites and its initiation codon, and in the latter, the manipulation is necessary to remove the stop codon of the first gene and to adjust the frames of the two genes. When two genes driven by the independent promoters are inserted into the E1 site, they might interfere with each other. However, when two independent expression units are inserted in different sites in the AdV genome, no interference occurs. Moreover, the advantage of this approach is that the main target gene can easily be changed using the AdV cassette that already contains the reporter gene.
There are three insertion sites and two orientations: a transgene can be inserted into the AdV genome by substitution of the E1 or E3 gene and by simple insertion at a position upstream of the E4 gene. Therefore, there are six different possible sites/orientations for any given transgene. Moreover, not only the potent promoters such as EF1 but also tissue-specific promoters such as -fetoprotein (AFP) can also be employed. Although the studies examining which sites/orientations are superior to others are practically important, they have been very limited11, 12 and systematic analyses have not been reported so far.
As it is known that the expression level of a transgene varies considerably depending on the site in the cell chromosome of the human genome, the phenomenon is called the position effect.13, 14 Although CG-methylation in the cell chromosome is clearly one reason, it is not observed in the AdV genome. Therefore, it would be of interest to examine whether the position effect might also be observed similarly in the AdV genome for the potent promoter and for the tissue-specific promoter.
FG AdVs retain almost all viral genes. They are normally not expressed in the target cells, because E1A protein, the essential transactivator for expression of all other viral genes, is not present. However, there is one report of splicing of aberrant mRNAs from the inserted foreign genes to a viral gene.15 In this case, the aberrant mRNAs are transcribed by strong foreign promoters and produce transgene-viral gene fusion proteins, which elicit strong immune responses. However, it is not known whether the production of the aberrant gene product between the inserted transgene and viral gene is rare or not.
In this study, we examined the AdV titers and expression levels of an identical transgene inserted at the E1, E3 and E4 sites. We used three transgenes, namely, GFP, LacZ and Cre, and two promoters, namely, the potent EF1 promoter and the cancer-specific AFP promoter, and attempted to construct AdVs using all combinations, that is, 18 AdVs, and succeeded in constructing 17 of them. We found that insertion at the E1 and E4 sites yielded mostly high titers, whereas the one at the E3 yielded variable titers. Surprisingly, four aberrantly spliced mRNAs between the transgenes and viral genes were found in the vector obtained by insertion at the E3 site, which was probably the reason for the very low titers. As for the expression levels, clear differences were observed among the vectors obtained with insertion at the E1, E3 and E4 sites despite using the identical transgene, indicating that the position effect was certainly present for the AdV genome and that aberrant splicing may, at least in part, explain this effect. We also propose a strategy to avoid generation of the aberrantly spliced mRNAs.
We first examined whether the vector titers were influenced by the site/orientations of the transgenes containing a potent EF1 promoter. Towards this end, we attempted to construct six GFP-expressing (EF-GFP) and six LacZ-expressing (EF-LacZ) vectors in all possible combinations, that is, the E1, E3 and E4 insertion sites and the two orientations ( Figure 1), and measured the vector titers (Figure 2a) (hereinafter, the vectors will be designated as per the following; the vectors containing the GFP gene and LacZ gene at the E1 insertion site and in the left orientation shall be denoted as G-E1L and Z-E1L vectors, respectively). Among the GFP-expressing vectors, high titers were obtained for G-E1L, G-E3L, G-E4L and G-E4R vectors (Figure 2a, bars 1, 3, 5 and 6), while the titer for the G-E1R vector was lower (bar 2). Notably, the G-E3R vector, that is, vector with the GFP transgene inserted in the E3 site in the rightward orientation, could not be obtained despite three independent attempts (bar 4, denote ). Therefore, although exactly the same EF1-GFP expression unit was inserted in these vectors, the sites and orientations exerted considerable influence on the vector titers and even determined whether the vector was available or not. Similar results were obtained for vectors expressing LacZ: the titers of the Z-E1L, Z-E4L and Z-E4R vectors (bars 7, 11 and 12) were high, and that of the Z-E1R vector was also low (bar 8). However, the results of insertion at the E3 site differed for GFP and LacZ. The titer ratio of Z-E3L was significantly lower than that of G-E3L (compare bars 3 and 9, described later), and the Z-E3R vector was available, although its titer was extremely low (bar 10). Therefore, the GFP gene and LacZ gene themselves influenced the vector titers.
The FG AdV structures of six different site/orientations in all possible combinations. The box containing pro, gene and pA represents the expression unit and the arrows show the orientation of transcription. pro, EF1 and AFP promoter; gene, GFP, LacZ and Cre; pA, rabbit -globin polyadenylation signal. For example, the vector containing the transgene at the E1 insertion site and in the left orientation is denoted as E1L.
Titers of the virus vectors containing identical expression units. (a) Virus titers of the AdVs containing the EF1 promoter. The AdV genomes transduced into the HuH-7 cells were measured 3 days post infection. The virus titers were calculated relative to the copy numbers of the AdVs.16 The titer of the E1L vector was set as 1; G-E1L, 8.3 108 relative virus titer (rVT)/ml, L-E1L, 5.0 109 rVT/ml. indicates that G-E3R could not be obtained. (b) The titers of the virus vector containing Cre gene driven by the AFP promoter. E1L vector was used as the control. *P<0.05, **P<0.01.
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Juvenile Arthritis report gene therapy approach
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An exhaustive hereditary analysis of a large Louisiana family with vision issues has uncovered a new gene tied to an incurable eye disorder called retinitis pigmentosa, according to an examination led by scientists at The University of Texas Health Science Center at Houston (UTHealth). It is a family of eye diseases that affects more than 200,000 in the United States and millions worldwide
The retina converts images into electrical signals that can be processed by the brain. It acts much like the film in a camera. Retinitis pigmentosa damages this film (the retina) and its early symptoms include decreased night vision and peripheral vision. Once it starts, the loss of vision is relentlessly progressive, often ending in blindness.
In the journal Investigative Ophthalmology & Visual Science, UTHealth's Stephen P. Daiger, Ph.D., and his colleagues report their discovery of a new gene tied to retinitis pigmentosa, which brings the total of genes associated with this sight-threatening disease to more than 60. The gene is called hexokinase 1 (HK1).
This information is important because it helps affected families cope with the disorder, helps explain the biologic basis of these diseases and suggests targets for drug treatments and gene therapy, said Daiger, the report's senior author and holder of the Thomas Stull Matney Ph.D. Endowed Professorship in Environmental and Genetic Sciences at UTHealth School of Public Health.
"The challenge now is to block the activity of these mutations and clinical trials are underway to do just that," he said.
"Dr. Daiger is trying to make a breakthrough in potentially blinding diseases with no known treatments," said Richard S. Ruiz, M.D., professor of ophthalmology and holder of the John S. Dunn Distinguished University Chair in Ophthalmology at UTHealth. "Right now, we address the symptoms of the disease and help patients make the most of their existing vision."
For approximately three decades, Daiger, a member of the Human Genetics Center at the UTHealth School of Public Health, has been following the progress of hundreds of families across the country with retinitis pigmentosa. "We've found the cause of disease in 80 percent of the families we have studied," Daiger said. "Our goal is to find the cause in the remaining 20 percent."
Equipped with the genetic profiles of family members, Daiger's team has identified differences in the genetic makeup of those with the disease. The researchers also use family histories and DNA tests to glean information about the condition's hereditary nature.
There are different types of retinitis pigmentosa and Daiger's laboratory is focused on the autosomal dominant type. This means that only one parent needs the mutation in order to pass the disease to a child. This type accounts for about a third of all cases and many of its disease-causing genes have been discovered, several by Daiger's research group.
"The story of the HK1 mutation is itself interesting. What we found is a mutation present in families from Louisiana, Canada and Sicily. Our evidence suggests the mutation arose in a common ancestor who lived centuries ago," Daiger said. "The mutation spread in Europe and North America, and may be common among Acadians in Louisiana. This is called a founder mutation."
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Worlds First Gene Therapy Trial To Cure Deafness - Nov 2014
DENVER - A local man is a making medical history. Rob Gerk is the first person in the world to receive gene therapy to restore his hearing. "It was very inte...
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Worlds First Gene Therapy Trial To Cure Deafness - Nov 2014 - Video
Gene Therapy (2015) 22, 2028; doi:10.1038/gt.2014.101; published online 6 November 2014
K LJackson1, R DDayton1, E AOrchard2, SJu3, DRinge4, G APetsko4,5, L EMaquat6,7 and R LKlein1
Amyotrophic lateral sclerosis (ALS) is a deadly neurodegenerative disease involving progressive paralysis. There are no highly efficacious strategies to treat ALS despite great effort by doctors and scientists. Successful treatments in mouse models, most of which are based on rare familial mutations in the ALS gene SOD1, have so far had little impact on modifying the disease in humans. Novel models based on transactive response DNA-binding protein 43kDa (TDP-43) may offer a more predictive test system given that the vast majority of ALS cases harbor TDP-43 pathology in their neurons and glia.1, 2, 3 Abnormal TDP-43 aggregates are also prominent in the class of diseases known as frontotemporal lobar degeneration (FTLD-TDP).4 TDP-43 is an RNA-binding protein that is normally found predominantly in the nucleus. In FTLD-TDP and the majority of ALS, abnormal TDP-43 accumulation occurs in the cytoplasm in the form of hyperphosphorylated and ubiquitinated pathological protein aggregates, and thus serves as a post-mortem diagnostic marker. 1, 2, 3, 4 One of the ways by which TDP-43 has been studied in animals is by gene delivery, which has proven to be sufficiently reproducible to allow the discrimination of genotypephenotype differences among TDP-43 isoforms in our previous work.5, 6, 7 This reproducibility and the ability to experimentally control the onset and severity of the disease state offer advantages for modeling, given that TDP-43 overexpression is highly toxic to cells.8 Here we use TDP-43 gene transfer to induce motor paralysis in rats to study limb symptomatology that is germane to ALS as a platform for gene therapy. Overexpression of TDP-43 causes progressive paresis to paralysis of the limbs in a highly reproducible manner,6, 7 offering an assay for therapeutic efficacy such as gene therapy. Gene therapy is worth considering for this disease given that ALS is fatal and irreversible. In this report, recombinant TDP-43 expression was titrated to a low level for a partial disease state retaining restorative capacity.
Refinement of TDP-43 animal models continues to be an important goal in the field.9 Reports of experimental treatments that slow or block TDP-43-mediated toxicity are beginning to emerge, either by genetic or pharmacological interventions in several TDP-43 models.10, 11, 12, 13, 14 Here we report behavioral outcomes from testing an empirically chosen therapeutic target, cDNA coding for human upframeshift protein 1 (UPF1), in a rat model of ALS-like paralysis based on TDP-43.
UPF1 is best known for its role in nonsense-mediated mRNA decay (NMD), a surveillance mechanism that degrades mRNAs containing a premature termination codon, which can be generated, for example, through alternative splicing. NMD prevents the production of truncated proteins that could harm the cell. NMD is also involved in the regulation of the expression of ~10% of normal physiologic transcripts in the cell, and is essential in mice.15, 16, 17 We pursued the possibility that UPF1 could ameliorate ALS-like symptoms based on the work carried out in yeast and neuronal cultures.18, 19 In a genetic screen of several thousands of proteins, a yeast homolog of hUPF1, and then the human gene itself, was found to prevent FUS- and TDP-43-mediated toxicity in yeast, 19 Ju et al., unpublished. Barmada et al.18 have advanced this approach, demonstrating that UPF1 protects primary neuronal cultures from TDP-43 cytotoxicity, possibly by upregulating NMD, as inhibitors of NMD blocked the protective effect. 18 The fact that expressing UPF1 blocks the toxic actions of TDP-43 in yeast cells and cultured neurons is consistent with the hypothesis that TDP-43-induced toxicity involves inhibition of UPF1 function, because TDP-43 toxicity can be suppressed by adding back UPF1 to restore NMD.
The main purpose of this study was to evaluate the expression of human mycUPF1 (i.e. human UPF1 with an N-terminal myc epitope tag) as a protection against TDP-43-induced limb paralysis in rats. MycUPF1 was tested in parallel with several different types of control treatments, all confirming that mycUPF1 elicits a specific therapeutic effect. We also evaluated whether the expression of recombinant TDP-43 or mycUPF1 would affect either each others recombinant gene expression or the expression of endogenous rat TDP-43 or UPF1. The data demonstrate that augmenting the cellular abundance of UPF1 provides a useful means of abrogating the devastating paralysis induced by TDP-43 overexpression.
Exogenous TDP-43 and green fluorescent protein (GFP) expression levels were purposefully set relatively low compared with the previous studies to test a rat model with a partial lesion and restorative capacity. This titration was advantageous to observe a therapeutic effect, but the low expression levels rendered detection of the transgene products inefficient. Nevertheless, previous work demonstrated that intravenous adenoassociated virus vector (AAV9) TDP-43 gene transfer specifically induces hindlimb paralysis even when the resulting level of exogenous TDP-43 is only faintly detectable.6 We chose the intravenous AAV9 method because it produces widespread central nervous system (CNS) expression, leading to marked expression in spinal motor neurons, dorsal root ganglia (DRG) neurons and cerebellar Purkinje neurons,6, 20 with only a small fold overexpression of the encoded protein, for example, less than twofold overexpression relative to the corresponding endogenous protein as estimated in the spinal cord in Dayton et al.7
For studying the effect of mycUPF1 expression, we harvested DRG neurons because this tissue provides a relatively high percentage of transduced cells in the nervous system, allowing for detection of transgene product. By comparison, the spinal cord and cerebellar samples include a greater percentage of non-transduced cells. We used antibodies for total TDP-43 or total UPF1 that detect both the endogenous rat plus exogenous human TDP-43 or UPF1. In DRG, the increase in total TDP-43 expression in AAV9 TDP-43/Empty vs uninjected animals was 2.4-fold (t-test, P<0.02, N=3 per group), whereas for total UPF1, we estimated the increase to be 1.6-fold in AAV9 mycUPF1 vs uninjected subjects ( Figure 1). The fold increases were relatively lower in the spinal cord (Figure 1) and cerebellum (not shown), as expected: the estimated ratio in the spinal cord and cerebellum was 1.4- and 1.2-fold for AAV9 TDP-43/Empty vs uninjected subjects and 1.1- and 1.1-fold for AAV9 mycUPF1 vs uninjected subjects (N=3 per group). Although fold overexpression levels were small, recombinant mycUPF1 could be specifically visualized using myc antibody, which detected recombinant mycUPF1 only in subjects receiving AAV9 mycUPF1 only or AAV9 TDP-43/mycUPF1 ( Figure 2).
Overexpression of TDP-43 or UPF1 in the rat CNS. Protein from dissected DRG and lumbar spinal cord was analyzed by western blotting 12 weeks after intravenous injection of AAV9 expression vectors. Three animals are shown for each condition. The level of total TDP-43 (endogenous rat plus recombinant human TDP-43) was significantly increased in the DRG of the AAV9 TDP-43/Empty group compared with uninjected subjects (t-test, P<0.02, N=3), but less so in the spinal cord or cerebellum (not shown). The expression level of human mycUPF1 compared with endogenous rat UPF1 was relatively small in all the three regions. The bands were normalized to GAPDH. See Results for details.
Selective detection of only recombinant human TDP-43 or mycUPF1. (a) A human-specific TDP-43 antibody detected exogenous human but not endogenous rat TDP-43 in DRGs. The level of TDP-43 expression was indistinguishable with or without mycUPF1 coexpression. (b) The level of exogenous mycUPF1 was detected with a myc antibody and only observed in rats that received AAV9 mycUPF1. In contrast to (a), MycUPF1 expression levels were reduced when AAV9 TDP-43 was coexpressed (t-test, P<0.05, N=3 for DRG). The bands were normalized to GAPDH.
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Gene Therapy - Preservation of forelimb function by UPF1 ...
Gene therapy for cancer and other diseases
The recent two decades have seen dramatic progress in gene therapy, both scientifically and socially. We are now able to treat several single gene disorders,...
By: Oxford University Department for Continuing Education
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Gene Therapy To Restore Hearing Loss - Nov 2014
KANSAS CITY, Kan. Restoring hearing with a drug is the goal of a metro researcher. Now he #39;s begun the first study in humans of a gene therapy that could he...
By: sixesfullofnines
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HOPE, HYPE REALITY: SCIENTIFIC RATIONALE FOR GENOME EDITING
Why we consider gene therapy to be a viable means to control or cure HIV.
By: defeatHIV
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HOPE, HYPE & REALITY: SCIENTIFIC RATIONALE FOR GENOME EDITING - Video
Gene Therapy (2015) 22, 8795; doi:10.1038/gt.2014.85; published online 18 September 2014
Autosomal dominant familial hypercholesterolemia (FH) is caused by mutations in the low-density lipoprotein receptor (LDLR).1 Homozygous FH patients present with massively elevated LDL cholesterol (LDL-C) and cardiovascular disease. They have severe atherosclerosis and die of ischemic heart disease usually in their third decade of life. The majority of homozygous and a substantial proportion of heterozygous patients are refractory to conventional pharmacological therapy. Therapeutic options for these resistant patients are limited to LDL apheresis, portacaval anastomosis or liver transplantation.2 Gene therapy has been explored as an alternative treatment. Liver is the main target organ for FH gene therapy because of its capacity to dispose excess cholesterol by diverting it into bile acids; it is also accessible to gene delivery via the intravenous (i.v.) route or the hepatic artery. A number of studies have shown that hepatic reconstitution of LDLR expression ex vivo can reverse hypercholesterolemia, including promising results in a rabbit model of FH. 3 In the only clinical gene therapy trial for FH to date, Grossman et al.4, 5 isolated hepatocytes from FH patients, transduced them ex vivo with retroviral vector expressing LDLR and reimplanted them into the liver of the patients. Only marginal therapeutic benefit was achieved in this study. It was difficult to determine whether the reduction in LDL-C level was the direct result of the gene transfer or other factors were involved. Plasma LDL level is determined by LDL production and removal. For example, the decline of LDL-C after portacaval anastomosis is caused by a decreased secretion of very-low-density lipoprotein, a precursor of LDL, not by an enhanced LDL removal.6 In this clinical trial, LDL turnover was not measured, which led to the comment a modest 17% fall in plasma cholesterol after 25% hepatectomy and re-infusion of hepatocytes infected with a retrovirus might have been due to either diminished lipoprotein production or to enhanced activity of the patients own receptor.7 The focus has shifted to in vivo gene therapy thereafter. Helper-dependent adenoviral vector (HDAd) is devoid of all viral protein genes and has shown considerable promise for liver-directed gene transfer with long-term transgene expression, which lasted a lifetime in mice.8 In a previous study in LDLR/ mice, we showed that a single injection of HDAd expressing monkey LDLR reduced plasma cholesterol over 2 years and attenuated atherosclerotic lesion progression. 9 We also demonstrated that LDLR gene therapy induces the regression of established atherosclerosis in LDLR/ mice.10 Despite promising results of gene therapy in small animal models, its efficacy in large animal models has not been tested; there are important differences in physiology and in immune responses between rodents and humans. This issue is particularly relevant in gene therapy for lipid disorders.11
A nonhuman primate model of FH has been described in rhesus monkeys,12, 13 which carried a heterozygous nonsense mutation involving codon Trp283 14 of the LDLR. Extensive cross-breeding of the affected monkeys failed to yield any homozygotes, indicating that the mutation may be linked to a lethal mutation. With the availability only of the heterozygous (LDLR+/) rhesus monkey, we will be modeling heterozygous FH in humans, a relatively common genetic disorder that affects about 1 in 500 people in most ethnic groups.15 Heterozygous LDLR-deficient monkeys displayed elevated plasma cholesterol (5.176.47mmoll1 or 200250mgdl1) compared with unaffected monkeys (2.593.36mmoll1 or 100130mgdl1); the plasma cholesterol level further increased to 12.9320.69mmoll1 (500800mgdl1) when the animals were fed a high-cholesterol diet.16 In this study, we tested the efficacy of HDAd-based monkey LDLR gene therapy in high-cholesterol diet-fed LDLR+/ rhesus monkeys. We compared the effect of i.v. injection of HDAd-LDLR with that of a balloon catheter-based procedure developed by Brunetti-Pierri et al. 17 We found that a single i.v. injection of HDAd-LDLR into LDLR+/ monkeys produced a >50% lowering of plasma cholesterol that lasted about a month. We next tested a modified percutaneous catheter-based gene delivery strategy also developed by Brunetti-Pierri et al. 18 In this refinement, the HDAd-LDLR was injected directly into the hepatic artery in the presence of increased intrahepatic pressure induced by transient blockage of hepatic venous drainage by a balloon catheter positioned in the inferior vena cava (IVC). The optimized gene delivery strategy was highly efficacious in reducing the vector dose while substantially prolonging the therapeutic hypocholesterolemic response to the treatment regimen.
We treated four LDLR+/ monkeys as study subjects with a single i.v. injection of escalating doses of HDAd-LDLR. 9 We first treated monkey #8796 with 20ml of saline and found no significant changes in plasma cholesterol levels after treatment (Figure 1). As expected, we also failed to detect any change in plasma cholesterol when we treated another LDLR+/ monkey #9908 with an empty vector HDAd-0 (0.8 1012 viral particles (vp)kg). We next injected i.v. HDAd-LDLR into a third LDLR+/ monkey #7139 at a dose of 1.1 1012vpkg1, an HDAd dose that is 10-fold higher than the dose of HDAd--fetoprotein that stimulated significant elevation in -fetoprotein secretion in serum in baboons,17 and again failed to observe any change in plasma cholesterol level. We then treated a fourth monkey #13090 at an even higher i.v. dose of 5 1012vpkg1 of HDAd-LDLR. The treatment was well tolerated by the monkey and led to a 60% reduction in plasma cholesterol from a baseline of 14.95mmoll1 (578mgdl1) to 5.90mmoll1 (229mgdl1) on day 7. The plasma cholesterol lowering persisted until day 21, when it went up to 10.70mmoll1 (413mgdl1) on day 28, and toward pre-treatment levels on day 42. These results indicate that a dose higher than 1.1 1012vpkg1 was needed to reverse hypercholesterolemia in LDLR+/ monkeys, and a dose of 5 1012vpkg1 significantly restored normal plasma cholesterol in a heterozygous FH monkey, an effect that lasted for about a month. We next treated a fifth monkey #11226 with an even higher dose of 8.4 1012vpkg1, which was modestly below a dose that had previously proven to be lethal, 19 and observed severe acute toxicity and lethality within a day of treatment. The clinical picture and necropsy revealed hemorrhagic shock syndrome likely resulting from the high dose of HDAd vector used.
Efficacy of intravenous injection of HDAd expressing monkey LDLR in heterozygous LDLR-deficient rhesus monkeys. Four heterozygous LDLR-deficient monkeys were treated with a single intravenous injection of saline (#8796), empty vector at a dose of 0.8 1012vpkg1 (#9908) or HDAd-LDLR at a dose of 1.1 1012vpkg1 (#7139) or 5 1012vpkg1 (#13090). Baseline cholesterol levels were 18.0mmoll1 (696mgdl1) in monkey #8796, 9.5mmoll1 (368mgdl1) in monkey #9908, 8.0mmoll1 (308mgdl1) in monkey #7139 and 15.0mmoll1 (578mgdl1) in monkey #13090. The broken line shows pre-treatment cholesterol levels.
To improve on i.v. vector injection as a delivery method, Brunetti-Perri et al. developed a protocol 17, 18 to deliver the vector via an intrahepatic arterial catheter. Simultaneously, under fluoroscopic guidance, they inserted a balloon catheter into the IVC via the femoral vein and positioned it over the hepatic venous outflow (Figure 2a). Intrahepatic arterial HDAd injection when the balloon was inflated led to a 10-fold increase in efficiency in transgene expression ( Figures 2b and c). The IVC occlusion was also monitored by the venous pressure (Figure 2d). We performed the same procedure in rhesus monkeys and injected the HDAd vector (2ml) within a minute via a hepatic artery catheter immediately after the balloon was inflated.
Balloon catheter-based hepatic artery injection. (a) Schematic diagram of hepatic artery injection. Liver circulation is isolated by inserting a balloon catheter via the femoral vein and placing it in the IVC. A second intra-arterial catheter is inserted into the hepatic artery through the contralateral femoral artery. The placement of the catheter is visualized using fluoroscopy. Once occlusion of the hepatic circulation has been established via the balloon catheter in the IVC, the vector is injected via the arterial catheter. The occlusion is confirmed by monitoring hepatic venous pressure through the third catheter inserted into the femoral vein. BD, bile duct; HA, hepatic artery; HV, hepatic vein; PV, portal vein. (b) Fluoroscopy image to confirm the position of a balloon catheter. (c) Fluoroscopy after the balloon inflated. Contrast reagent was injected to confirm that the catheter was placed at the IVC. (d) Venous pressure. Occlusion was monitored by venous pressure.
The monkeys used for this procedure are summarized in Table 1. We first performed the procedure in a chow-fed (Purina LabDiet5LEO, St Louis, MO, USA) normal LDLR+/+ (#19254) and a heterozygous LDLR+/ (#19499) monkey. The injection was done immediately after the balloon was deflated but while hepatic venous pressure remained high. As reported previously, 17,18 systemic blood pressure fell significantly when the balloon was inflated. We found that serum interleukin (IL)-6 level increased 30min after injection and peaked at 2h ( Figure 3a) but decreased to non-detectable levels by 72h. The procedure also led to transient and inconsistent changes in plasma liver enzymes ( Figures 3b and c). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels peaked at about 24h; the increase was mild and resolved by day 5. Plasma total cholesterol levels in the LDLR+/ (#19499) monkey decreased from a baseline of 5.70mmoll1 (219mgdl1) to 3.90mmoll1 (150mgdl1) within 24h. It gradually went back up over the next few days returning to baseline by day 5. The plasma cholesterol level did not change in the non-FH (LDLR+/+) (#19254) monkey ( Figure 3d).
Acute toxicity measurements associated with balloon catheter-based hepatic artery injection. One normal LDLR+/+ (#19254) and one heterozygous LDLR+/ (#19499) monkeys on normal chow were treated by an injection of saline and a complete blood test and IL-6 measurement were performed. (a) Plasma IL-6 levels. (b) Serum ALT levels. (c) Serum aspartate aminotransferase (AST) levels. (d) Plasma cholesterol levels.
We next fed monkeys with a rhesus Western diet (Texas Biomedical Research Institute, San Antonio, TX, USA) for 7 weeks before treatment and were kept on the diet afterward. We injected HDAd-LDLR (2 1012vpkg1) into four monkeys immediately after the balloon was deflated. The plasma cholesterol did not change in two wild-type LDLR+/+ monkeys (#19360 and #21588) suggesting that the gene delivery does not have an effect on the cholesterol dynamics in monkeys that express normal amounts of LDLR. Of the two heterozygous LDLR+/ monkeys, one (#19251) showed no change in plasma cholesterol ( Figure 4a, green line), whereas another LDLR+/ monkey (#19498) exhibited a 57% drop in plasma cholesterol level from 8.15mmoll1 (315mgdl1) to 3.25mmoll1 (126mgdl1) at day 7 ( Figure 4a, red line). So there was a heterogeneous response in heterozygous FH monkeys treated at this dose of HDAd-LDLR. The cholesterol-lowering effect of HDAd-LDLR in the LDLR+/ (#19498) monkey that responded to the treatment was sustained for about 100 days. The plasma-lowering effect reached its nadir 7 days, and stayed at or near the nadir for another 3 weeks. Afterward, it gradually rose to 5.09mmoll1 (197mgdl1) at day 78, and then to above the pre-treatment level (9.30mmoll1 or 361mgdl1) by day 105 ( Figure 4a, red line). The two wild-type LDLR+/+ monkeys maintained normal serum ALT throughout the observation period of 120 days. The LDLR+/ monkey (#19251) that did not show a hypocholesterolemic response also maintained normal ALT levels for 67 days, end of the observation period for this monkey. In contrast, the serum ALT of the LDLR+/ monkey (#19498) that showed a hypocholesterolemic response maintained a normal ALT level during the first 3 weeks of treatment when the plasma cholesterol showed an excellent response ( Figure 4a, red line). ALT began to edge above normal to 70Ul1 on day 36, and continued to go up to peak at 144Ul1 on day 72, before it started trending down, eventually returning to normal on day 120 ( Figure 4b, red line). It is noteworthy that this monkey that had responded to the treatment developed liver enzyme elevation late, and the delayed increase in serum ALT coincided with the onset of loss of the cholesterol-lowering effect of the treatment. Although the significance of the timing is unclear, we note that a similar pattern is evident in an experiment involving another LDLR+/ monkey (#19269, see below).
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Drugmakers have a slew of treatments for afflictions related to sex and drugs. Now they may have one for rock n roll.
Novartis AG (NOVN) is developing a gene therapy that may reverse hearing loss by stimulating the regrowth of microscopic hair cells in the inner ear, allowing people to hear. The hairs are destroyed by prolonged exposure to loud noise, and dont take root again naturally. Novartis treated the first patient in October after successful tests on rats.
While hearing loss is most common in the elderly, rates are high in the music industry and the military, and rising among teenagers who listen to music at high volume. Almost 13 percent of children and adolescents under 19 in the U.S. have permanent damage caused by excessive exposure to noise, according to the Centers for Disease Control and Prevention.
A little too much Lady Gaga, said Mark Fishman, the head of Novartis Institutes for BioMedical Research, which is developing the therapy. About 36 million people in the U.S. have some form of hearing loss, according to the Basel, Switzerland-based company.
A solution could mean big money for Novartis and GenVec Inc. (GNVC), its partner in developing the drug. Global sales of hearing aids and cochlear implants may reach a combined $9.5 billion globally by 2020, according to San Francisco-based Grand View Research, which provides information on industries including technology and health care.
Global sales of hearing aids and cochlear implants may reach a combined $9.5 billion globally by 2020, according to San Francisco-based Grand View Research, which provides information on industries including technology and health care. Close
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Global sales of hearing aids and cochlear implants may reach a combined $9.5 billion globally by 2020, according to San Francisco-based Grand View Research, which provides information on industries including technology and health care.
Novartis plans to test its treatment on 45 patients in the U.S., with results expected by 2017, according to a description of the trial on clinicaltrials.gov, the National Institutes of Healths database of studies. Its too early to say when the treatment might be approved, Fishman said.
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Lady Gaga at High Volume Drives Hearing-Loss Drug Search: Health