This document summarizes an undergraduate thesis project that used the genetic design platform Genome Compiler to engineer adeno-associated viral (AAV) vectors for treating different forms of Leber congenital amaurosis (LCA). The student designed AAV vectors for 12 different types of LCA defined by mutations in various genes, using the published design of Jacobson et al for LCA2 as a template. Figures 1-12 show the different AAV vectors designed in Genome Compiler for each type of LCA. The goal was to engineer the vectors in silico to ensure efficiency and plausibility before testing in animal models.
This PPT includes the details about some cardiovascular diseases and how they are treated using Gene Therapy. It also discuss about the vectors that are used in the process.
The concept of transferring genes to tissues for clinical applications has been discussed for nearly half a century, but the ability to manipulate genetic material via recombinant DNA technology has brought this goal to reality. ‘Gene Therapy’ covers both the research and clinical applications of the new genetic therapy techniques currently being developed. The application of molecular biology has revolutionized researchers understanding of many diseases and has been readily applied for diagnostic purposes. Now-a-day this is originally conceived as a way to treat life-threatening disorders (inborn errors, cancers) refractory to conventional treatment, gene therapy now is considered for many non–life-threatening conditions, including those adversely affecting a patient’s quality of life. The lack of suitable treatment has become a rational basis for extending the scope of gene therapy. It is not very far, the justifiable optimism that with increased biotechnological improvement, gene therapy will become a standard part of clinical practice.
This document discusses genomic technologies that can be used to observe the human genome and their applications. It covers microarrays, next-generation sequencing, DNA methylation, copy number variation, and more. Challenges include the cost of these technologies and integrating the large amounts of data they produce to improve healthcare.
Genetic engineers are developing new techniques to turn cells into miniature drug factories by delivering synthetic DNA. This is done using either viral or plasmid vectors to transport the DNA past the cell's lipid bilayer and into the nucleus. Techniques like electroporation, gene guns, and novel injection devices use physical forces to disrupt the bilayer and allow DNA entry. Team is working with ChronTech Pharma to optimize a new injection device called IVIN that uses an array of needles to inject DNA vaccines into muscle tissue with improved transfection results. Overcoming the challenge of efficiently delivering genetic material into cells opens possibilities for treating previously untreatable diseases.
CRISPR/Cas9 is a new genome editing tool that allows geneticists to precisely edit DNA sequences. It uses a bacterial immune system to cut DNA at a specific location so that parts of the genome can be removed, replaced, or added. The system involves a Cas9 enzyme guided by RNA to a targeted DNA site, where it cuts both strands of the DNA. This allows desired modifications to the genome. The summary discusses how CRISPR can be used to create genetic variability, develop disease resistance in crops, and potentially edit human organs for transplantation with less immune rejection risk. It also provides an example of using CRISPR to enhance blast resistance in rice.
A simple version of the CRISPR/Cas system, CRISPR/Cas9, has been modified to edit genomes. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added.
Next generation sequencing (NGS) provides a high-throughput and cheaper alternative to DNA sequencing through massively parallel sequencing of millions of DNA fragments simultaneously. NGS can be used for target sequencing to identify disease-causing mutations, RNA sequencing to study entire transcriptomes, and has various applications in cancer research and treatment including identifying mutations that predict responses to immunotherapy. However, NGS also faces challenges like accurately sequencing regions with repeats and detecting fusion genes.
This document summarizes a seminar presentation on mixotrophy in land plants. Mixotrophy refers to the dual capability of both photosynthesis and organic carbon uptake. The presentation discusses the evolutionary pathways and diversity of mixotrophy strategies across eukaryotes. Tools for studying mixotrophy like isotopic tracing and genomic analysis are also reviewed. A case study examining the nuclear genomes of three mycoheterotrophic plants found profound reductions in photosynthesis and plastid-related genes, as well as increased substitution rates, demonstrating convergent evolution to heterotrophy.
This PPT includes the details about some cardiovascular diseases and how they are treated using Gene Therapy. It also discuss about the vectors that are used in the process.
The concept of transferring genes to tissues for clinical applications has been discussed for nearly half a century, but the ability to manipulate genetic material via recombinant DNA technology has brought this goal to reality. ‘Gene Therapy’ covers both the research and clinical applications of the new genetic therapy techniques currently being developed. The application of molecular biology has revolutionized researchers understanding of many diseases and has been readily applied for diagnostic purposes. Now-a-day this is originally conceived as a way to treat life-threatening disorders (inborn errors, cancers) refractory to conventional treatment, gene therapy now is considered for many non–life-threatening conditions, including those adversely affecting a patient’s quality of life. The lack of suitable treatment has become a rational basis for extending the scope of gene therapy. It is not very far, the justifiable optimism that with increased biotechnological improvement, gene therapy will become a standard part of clinical practice.
This document discusses genomic technologies that can be used to observe the human genome and their applications. It covers microarrays, next-generation sequencing, DNA methylation, copy number variation, and more. Challenges include the cost of these technologies and integrating the large amounts of data they produce to improve healthcare.
Genetic engineers are developing new techniques to turn cells into miniature drug factories by delivering synthetic DNA. This is done using either viral or plasmid vectors to transport the DNA past the cell's lipid bilayer and into the nucleus. Techniques like electroporation, gene guns, and novel injection devices use physical forces to disrupt the bilayer and allow DNA entry. Team is working with ChronTech Pharma to optimize a new injection device called IVIN that uses an array of needles to inject DNA vaccines into muscle tissue with improved transfection results. Overcoming the challenge of efficiently delivering genetic material into cells opens possibilities for treating previously untreatable diseases.
CRISPR/Cas9 is a new genome editing tool that allows geneticists to precisely edit DNA sequences. It uses a bacterial immune system to cut DNA at a specific location so that parts of the genome can be removed, replaced, or added. The system involves a Cas9 enzyme guided by RNA to a targeted DNA site, where it cuts both strands of the DNA. This allows desired modifications to the genome. The summary discusses how CRISPR can be used to create genetic variability, develop disease resistance in crops, and potentially edit human organs for transplantation with less immune rejection risk. It also provides an example of using CRISPR to enhance blast resistance in rice.
A simple version of the CRISPR/Cas system, CRISPR/Cas9, has been modified to edit genomes. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added.
Next generation sequencing (NGS) provides a high-throughput and cheaper alternative to DNA sequencing through massively parallel sequencing of millions of DNA fragments simultaneously. NGS can be used for target sequencing to identify disease-causing mutations, RNA sequencing to study entire transcriptomes, and has various applications in cancer research and treatment including identifying mutations that predict responses to immunotherapy. However, NGS also faces challenges like accurately sequencing regions with repeats and detecting fusion genes.
This document summarizes a seminar presentation on mixotrophy in land plants. Mixotrophy refers to the dual capability of both photosynthesis and organic carbon uptake. The presentation discusses the evolutionary pathways and diversity of mixotrophy strategies across eukaryotes. Tools for studying mixotrophy like isotopic tracing and genomic analysis are also reviewed. A case study examining the nuclear genomes of three mycoheterotrophic plants found profound reductions in photosynthesis and plastid-related genes, as well as increased substitution rates, demonstrating convergent evolution to heterotrophy.
This document discusses recombinant DNA technology, which involves introducing foreign DNA into a host organism. It describes the basic steps of isolating DNA, cutting it with restriction enzymes, amplifying the gene with PCR, and ligating it into a vector for insertion into the host. The key tools used are restriction enzymes, DNA polymerase for PCR, ligase, and vectors to introduce the recombinant DNA into hosts such as bacteria. The goal is to generate organisms that produce desired proteins or have new traits.
1. The document discusses research into visualizing the interaction between the HIV viral envelope and capsid protein using freeze-fracture electron microscopy.
2. The goal is to better understand how the viral envelope attaches to the capsid, which could lead to methods to disrupt this process and inhibit HIV infection.
3. Introducing a premature stop codon in the Gag protein will arrest viral bud release, allowing visualization of the arrested viral buds and interaction between the envelope and capsid.
Gene therapy involves introducing genes into cells to treat or prevent disease. It works by correcting defective genes that cause disease or by making cells produce products to treat the disease. The first approved gene therapy treated a girl for ADA-SCID. There are two main approaches - in vivo therapy directly delivers genes into body cells, while ex vivo therapy transfers genes to cultured cells before reinsertion. Viral vectors like retroviruses and adenoviruses are often used due to their ability to deliver genes, but come with risks like insertional mutagenesis. Non-viral methods include physical methods like microinjection and chemical methods using liposomes. Gene therapy shows promise for diseases like cancer, cardiovascular disease, and neurological disorders.
Gene editing using CRISPR was originally discovered as a bacterial immune system that provides resistance to viruses. CRISPR uses specialized DNA sequences and associated Cas proteins to create targeted double-strand breaks in DNA, allowing modification of genomes. The technique has rapidly advanced due to its simplicity and versatility compared to prior tools. CRISPR holds promise for treating genetic diseases, transplantation, biotechnology, disease models, and more. It has become a widely used research tool with many companies and publications emerging around its applications.
This document summarizes safety considerations for DNA vaccines. It discusses safety issues related to the genetic elements of DNA vaccines, including the potential for antibiotic resistance gene transfer and germline integration. It also discusses safety concerns regarding the microbial production host, such as endotoxin production and genetic instability. The document proposes strategies to improve safety, including using replication origins with narrow host range, non-antibiotic selection markers, artificial promoter and signal sequences with low human homology, and gram-positive microbial hosts like Lactococcus lactis that do not produce endotoxins.
Adenoviral vectors are modified adenoviruses that can deliver genetic material into host cells. Adenoviruses are medium-sized, non-enveloped viruses containing double-stranded DNA. They can efficiently transfer DNA/RNA into cells and have been used to construct viral vectors. Wild type adenoviruses are modified by deleting non-essential genes and adding exogenous genetic material to create viral vectors. Three generations of adenoviral vectors have been developed for gene therapy, with later generations having improved safety profiles and ability to carry larger DNA payloads.
This slidedeck details two comprehensive informatics solutions — the Biomedical Genomics Workbench and Ingenuity Knowledge Base Variant Analysis platforms. We show the intuitive user interface of CLC Cancer Research Workbench and demonstrate how the rich biological content from Ingenuity Knowledge Base helps you rapidly identify critical variants in your samples.
This study evaluated the use of targeted next-generation sequencing (NGS) for preimplantation genetic diagnosis (PGD) of single-gene disorders. The study compared NGS results from embryo biopsies to results from two established PGD methods. NGS provided 100% consistency with the established methods in diagnosing point mutations and small insertions/deletions in six couples at risk of transmitting single-gene disorders. Additionally, NGS allowed for parallel single-gene disorder screening and comprehensive chromosome screening from the same biopsy sample. The study demonstrates NGS can provide accurate and consistent PGD results and could serve as a model for further development of this emerging technology in PGD.
This document discusses the evolution of next-generation sequencing (NGS) technologies over the past decade. It begins by describing how early NGS platforms enabled massively parallel sequencing through the clonal amplification of DNA templates on beads or solid surfaces. It then explains the two main approaches used in NGS - sequencing by ligation (SBL) and sequencing by synthesis (SBS) - and how they identify nucleotide sequences. The document evaluates the benefits and limitations of various NGS platforms and approaches.
CRISPR-Cas9 is a powerful gene editing tool that has promising applications in public health. It allows targeted editing of genes and could help treat diseases like HIV/AIDS, cancer, and antibiotic resistance. However, there are also ethical concerns about its use, such as off-target effects and questions around human enhancement. Going forward, CRISPR holds potential for developing new therapies and improving agriculture, but its applications will require addressing safety, consent, and access issues.
RT-PCR and DNA microarray measurement of mRNA cell proliferationIJAEMSJORNAL
For mRNA quantification, RT-PCR and DNA microarrays have been compared in few studies
(RT-PCR). Healing callus of adult and juvenile rats after femur injury was found to be rich in mRNA at
various stages of the healing process. We used both methods to examine ten samples and a total of 26 genes.
Internal DNA probes tagged with 32P were employed in reverse transcription-polymerase chain reaction
(RT-PCR) to identify genes (RT-PCR). Ten Affymetrix® Rat U34A cRNA microarrays were hybridized with
biotin-labeled cRNA generated from mRNA. There was a wide range of correlation coefficients (r) between
RT-PCR and microarray data for each gene. Meaning became genetically unique because of this diversity.
Relatively lowly expressed genes had the highest r values. The distance between PCR primers and
microarray probes was found to be higher than previously assumed, leading to a drop in agreement between
microarray calls and PCR outcomes. Microarray research showed that RT-PCR expression levels for two
genes had a "floor effect." As a result, PCR primers and microarray probes that overlap in mRNA expression
levels can provide good agreement between these two techniques.
Molecular approaches in improvement of fruit cropsShabnamSyed3
This document provides an overview of molecular markers and their applications in horticultural crop improvement. It begins with definitions of molecular markers and explains why they are useful tools. It then discusses different types of molecular markers including morphological, biochemical, and DNA-based markers. The document outlines several molecular techniques used for marker analysis, such as polymerase chain reaction (PCR), electrophoresis, hybridization, and DNA sequencing. It provides examples of how molecular markers can be used for genetic diversity analysis, quantitative trait locus (QTL) mapping, varietal identification, disease diagnostics, and marker-assisted selection (MAS) in fruit crop breeding. Finally, it discusses properties of ideal molecular markers and limitations of QTL mapping.
GTC group 8 - Next Generation SequencingYanqi Chan
DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. Discuss the application of next generation sequencing in cancer treatment.
De novo transcriptome assembly of solid sequencing data in cucumis melobioejjournal
As sequencing technologies progress, focus shifts towards solving bioinformatic challenges, of which sequence read assembly is the first task. In the present study, we have carried out a comparison of two assemblers (SeqMan and CLC) for transcriptome assembly, using a new dataset from Cucumis melo. Between two assemblers SeqMan generated an excess of small, redundant contigs where as CLC generated the least redundant assembly. Since different assemblers use different algorithms to build contigs, wefollowed the merging of assemblies by CAP3 and found that the merged assembly is better than individual assemblies and more consistent in the number and size of contigs. Combining the assemblies from different programs gave a more credible final product, and therefore this approach is recommended for quantitative
output.
This document discusses analytical validation needs for next-generation sequencing, including somatic variants. It notes that targeted sequencing and whole genome sequencing have similar analytical validation requirements but reference data needs to cover all genomic regions. There is limited utility in benchmarking or reference pipelines as custom assay development often uses custom informatics. Increased transparency is needed on exact intervals tested and validation metrics based on variant type and allelic fraction.
SBVRLDNACOMP:AN EFFECTIVE DNA SEQUENCE COMPRESSION ALGORITHMijcsa
There are plenty specific types of data which are needed to compress for easy storage and to reduce overall retrieval times. Moreover, compressed sequence can be used to understand similarities between biological sequences. DNA data compression challenge has become a major task for many researchers for the last few years as a result of exponential increase of produced sequences in gene databases. In this research paper we have attempt to develop an algorithm by self-reference bases; namely Single Base Variable Repeat Length DNA Compression (SBVRLDNAComp). There are a number of reference based compression methods but they are not satisfactory for forthcoming new species. SBVRLDNAComp is an optimal solution of the result obtained from small to long, uniform identical and non-identical string of nucleotides checked in four different ways. Both exact repetitive and non-repetitive bases are compressed by SBVRLDNAComp.The sound part of it is without any reference database BVRLDNAComp achieves 1.70 to 1.73 compression ratio α after testing on ten benchmark DNA sequences. The compressed file can be further compressed with standard tools (such as WinZip or WinRar) but even without this SBVRLDNAComp outperforms many standard DNA compression algorithms.
Gene therapy involves techniques that modify or manipulate genes to treat or prevent diseases. The first gene therapy treatment occurred in 1990 for severe combined immunodeficiency. There are four main approaches to gene therapy: inserting a normal gene to compensate for a defective one, replacing an abnormal gene with a normal one, repairing an abnormal gene, or altering gene regulation. Viruses are commonly used as vectors to deliver therapeutic genes into target cells, with retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses being some of the most widely used viral vectors, each with advantages and limitations.
An accurate distance_to_the_nearest_galaxySérgio Sacani
This document summarizes recent research that more accurately measured the distance to the Large Magellanic Cloud (LMC) galaxy. A team called the Araucaria Project measured the distances to eight eclipsing binary stars in the LMC to determine a distance of 49.3 ± 0.5 kiloparsecs, which is accurate to 2.2%. This more precise measurement of the distance to the LMC will help astronomers better determine distances to more distant galaxies and improve our understanding of properties like dark energy. The new measurement is consistent with but more accurate than previous estimates, addressing issues with prior discrepancies in LMC distance measurements.
This document provides an overview of gene therapy. It discusses the basic concept of replacing a mutated gene with a healthy copy to cure diseases. It describes the central dogma of molecular biology and barriers to gene therapy like developing effective carriers or vectors. The main types of vectors discussed are viral vectors, including RNA virus vectors like retroviruses and DNA virus vectors like adenoviruses and adeno-associated viruses. Clinical trials of gene therapy are also mentioned, with the first using retroviruses to treat severe combined immunodeficiency. In conclusion, the document states that gene therapy requires identifying target genes, developing safe and efficient vectors, and conducting clinical trials optimized for specific diseases.
On January 25, 2022, Nature published an article listing seven technologies worthy of attention this year. Targeted genetic therapies was on the list. The remaining six technologies are: Fully finished genomes, Protein structure solutions, Quantum simulation, Precise genome manipulation, Spatial multi-omics), CRISPR-based diagnostics.
This document discusses recombinant DNA technology, which involves introducing foreign DNA into a host organism. It describes the basic steps of isolating DNA, cutting it with restriction enzymes, amplifying the gene with PCR, and ligating it into a vector for insertion into the host. The key tools used are restriction enzymes, DNA polymerase for PCR, ligase, and vectors to introduce the recombinant DNA into hosts such as bacteria. The goal is to generate organisms that produce desired proteins or have new traits.
1. The document discusses research into visualizing the interaction between the HIV viral envelope and capsid protein using freeze-fracture electron microscopy.
2. The goal is to better understand how the viral envelope attaches to the capsid, which could lead to methods to disrupt this process and inhibit HIV infection.
3. Introducing a premature stop codon in the Gag protein will arrest viral bud release, allowing visualization of the arrested viral buds and interaction between the envelope and capsid.
Gene therapy involves introducing genes into cells to treat or prevent disease. It works by correcting defective genes that cause disease or by making cells produce products to treat the disease. The first approved gene therapy treated a girl for ADA-SCID. There are two main approaches - in vivo therapy directly delivers genes into body cells, while ex vivo therapy transfers genes to cultured cells before reinsertion. Viral vectors like retroviruses and adenoviruses are often used due to their ability to deliver genes, but come with risks like insertional mutagenesis. Non-viral methods include physical methods like microinjection and chemical methods using liposomes. Gene therapy shows promise for diseases like cancer, cardiovascular disease, and neurological disorders.
Gene editing using CRISPR was originally discovered as a bacterial immune system that provides resistance to viruses. CRISPR uses specialized DNA sequences and associated Cas proteins to create targeted double-strand breaks in DNA, allowing modification of genomes. The technique has rapidly advanced due to its simplicity and versatility compared to prior tools. CRISPR holds promise for treating genetic diseases, transplantation, biotechnology, disease models, and more. It has become a widely used research tool with many companies and publications emerging around its applications.
This document summarizes safety considerations for DNA vaccines. It discusses safety issues related to the genetic elements of DNA vaccines, including the potential for antibiotic resistance gene transfer and germline integration. It also discusses safety concerns regarding the microbial production host, such as endotoxin production and genetic instability. The document proposes strategies to improve safety, including using replication origins with narrow host range, non-antibiotic selection markers, artificial promoter and signal sequences with low human homology, and gram-positive microbial hosts like Lactococcus lactis that do not produce endotoxins.
Adenoviral vectors are modified adenoviruses that can deliver genetic material into host cells. Adenoviruses are medium-sized, non-enveloped viruses containing double-stranded DNA. They can efficiently transfer DNA/RNA into cells and have been used to construct viral vectors. Wild type adenoviruses are modified by deleting non-essential genes and adding exogenous genetic material to create viral vectors. Three generations of adenoviral vectors have been developed for gene therapy, with later generations having improved safety profiles and ability to carry larger DNA payloads.
This slidedeck details two comprehensive informatics solutions — the Biomedical Genomics Workbench and Ingenuity Knowledge Base Variant Analysis platforms. We show the intuitive user interface of CLC Cancer Research Workbench and demonstrate how the rich biological content from Ingenuity Knowledge Base helps you rapidly identify critical variants in your samples.
This study evaluated the use of targeted next-generation sequencing (NGS) for preimplantation genetic diagnosis (PGD) of single-gene disorders. The study compared NGS results from embryo biopsies to results from two established PGD methods. NGS provided 100% consistency with the established methods in diagnosing point mutations and small insertions/deletions in six couples at risk of transmitting single-gene disorders. Additionally, NGS allowed for parallel single-gene disorder screening and comprehensive chromosome screening from the same biopsy sample. The study demonstrates NGS can provide accurate and consistent PGD results and could serve as a model for further development of this emerging technology in PGD.
This document discusses the evolution of next-generation sequencing (NGS) technologies over the past decade. It begins by describing how early NGS platforms enabled massively parallel sequencing through the clonal amplification of DNA templates on beads or solid surfaces. It then explains the two main approaches used in NGS - sequencing by ligation (SBL) and sequencing by synthesis (SBS) - and how they identify nucleotide sequences. The document evaluates the benefits and limitations of various NGS platforms and approaches.
CRISPR-Cas9 is a powerful gene editing tool that has promising applications in public health. It allows targeted editing of genes and could help treat diseases like HIV/AIDS, cancer, and antibiotic resistance. However, there are also ethical concerns about its use, such as off-target effects and questions around human enhancement. Going forward, CRISPR holds potential for developing new therapies and improving agriculture, but its applications will require addressing safety, consent, and access issues.
RT-PCR and DNA microarray measurement of mRNA cell proliferationIJAEMSJORNAL
For mRNA quantification, RT-PCR and DNA microarrays have been compared in few studies
(RT-PCR). Healing callus of adult and juvenile rats after femur injury was found to be rich in mRNA at
various stages of the healing process. We used both methods to examine ten samples and a total of 26 genes.
Internal DNA probes tagged with 32P were employed in reverse transcription-polymerase chain reaction
(RT-PCR) to identify genes (RT-PCR). Ten Affymetrix® Rat U34A cRNA microarrays were hybridized with
biotin-labeled cRNA generated from mRNA. There was a wide range of correlation coefficients (r) between
RT-PCR and microarray data for each gene. Meaning became genetically unique because of this diversity.
Relatively lowly expressed genes had the highest r values. The distance between PCR primers and
microarray probes was found to be higher than previously assumed, leading to a drop in agreement between
microarray calls and PCR outcomes. Microarray research showed that RT-PCR expression levels for two
genes had a "floor effect." As a result, PCR primers and microarray probes that overlap in mRNA expression
levels can provide good agreement between these two techniques.
Molecular approaches in improvement of fruit cropsShabnamSyed3
This document provides an overview of molecular markers and their applications in horticultural crop improvement. It begins with definitions of molecular markers and explains why they are useful tools. It then discusses different types of molecular markers including morphological, biochemical, and DNA-based markers. The document outlines several molecular techniques used for marker analysis, such as polymerase chain reaction (PCR), electrophoresis, hybridization, and DNA sequencing. It provides examples of how molecular markers can be used for genetic diversity analysis, quantitative trait locus (QTL) mapping, varietal identification, disease diagnostics, and marker-assisted selection (MAS) in fruit crop breeding. Finally, it discusses properties of ideal molecular markers and limitations of QTL mapping.
GTC group 8 - Next Generation SequencingYanqi Chan
DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. Discuss the application of next generation sequencing in cancer treatment.
De novo transcriptome assembly of solid sequencing data in cucumis melobioejjournal
As sequencing technologies progress, focus shifts towards solving bioinformatic challenges, of which sequence read assembly is the first task. In the present study, we have carried out a comparison of two assemblers (SeqMan and CLC) for transcriptome assembly, using a new dataset from Cucumis melo. Between two assemblers SeqMan generated an excess of small, redundant contigs where as CLC generated the least redundant assembly. Since different assemblers use different algorithms to build contigs, wefollowed the merging of assemblies by CAP3 and found that the merged assembly is better than individual assemblies and more consistent in the number and size of contigs. Combining the assemblies from different programs gave a more credible final product, and therefore this approach is recommended for quantitative
output.
This document discusses analytical validation needs for next-generation sequencing, including somatic variants. It notes that targeted sequencing and whole genome sequencing have similar analytical validation requirements but reference data needs to cover all genomic regions. There is limited utility in benchmarking or reference pipelines as custom assay development often uses custom informatics. Increased transparency is needed on exact intervals tested and validation metrics based on variant type and allelic fraction.
SBVRLDNACOMP:AN EFFECTIVE DNA SEQUENCE COMPRESSION ALGORITHMijcsa
There are plenty specific types of data which are needed to compress for easy storage and to reduce overall retrieval times. Moreover, compressed sequence can be used to understand similarities between biological sequences. DNA data compression challenge has become a major task for many researchers for the last few years as a result of exponential increase of produced sequences in gene databases. In this research paper we have attempt to develop an algorithm by self-reference bases; namely Single Base Variable Repeat Length DNA Compression (SBVRLDNAComp). There are a number of reference based compression methods but they are not satisfactory for forthcoming new species. SBVRLDNAComp is an optimal solution of the result obtained from small to long, uniform identical and non-identical string of nucleotides checked in four different ways. Both exact repetitive and non-repetitive bases are compressed by SBVRLDNAComp.The sound part of it is without any reference database BVRLDNAComp achieves 1.70 to 1.73 compression ratio α after testing on ten benchmark DNA sequences. The compressed file can be further compressed with standard tools (such as WinZip or WinRar) but even without this SBVRLDNAComp outperforms many standard DNA compression algorithms.
Gene therapy involves techniques that modify or manipulate genes to treat or prevent diseases. The first gene therapy treatment occurred in 1990 for severe combined immunodeficiency. There are four main approaches to gene therapy: inserting a normal gene to compensate for a defective one, replacing an abnormal gene with a normal one, repairing an abnormal gene, or altering gene regulation. Viruses are commonly used as vectors to deliver therapeutic genes into target cells, with retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses being some of the most widely used viral vectors, each with advantages and limitations.
An accurate distance_to_the_nearest_galaxySérgio Sacani
This document summarizes recent research that more accurately measured the distance to the Large Magellanic Cloud (LMC) galaxy. A team called the Araucaria Project measured the distances to eight eclipsing binary stars in the LMC to determine a distance of 49.3 ± 0.5 kiloparsecs, which is accurate to 2.2%. This more precise measurement of the distance to the LMC will help astronomers better determine distances to more distant galaxies and improve our understanding of properties like dark energy. The new measurement is consistent with but more accurate than previous estimates, addressing issues with prior discrepancies in LMC distance measurements.
This document provides an overview of gene therapy. It discusses the basic concept of replacing a mutated gene with a healthy copy to cure diseases. It describes the central dogma of molecular biology and barriers to gene therapy like developing effective carriers or vectors. The main types of vectors discussed are viral vectors, including RNA virus vectors like retroviruses and DNA virus vectors like adenoviruses and adeno-associated viruses. Clinical trials of gene therapy are also mentioned, with the first using retroviruses to treat severe combined immunodeficiency. In conclusion, the document states that gene therapy requires identifying target genes, developing safe and efficient vectors, and conducting clinical trials optimized for specific diseases.
On January 25, 2022, Nature published an article listing seven technologies worthy of attention this year. Targeted genetic therapies was on the list. The remaining six technologies are: Fully finished genomes, Protein structure solutions, Quantum simulation, Precise genome manipulation, Spatial multi-omics), CRISPR-based diagnostics.
Gene therapy is a medical technology which aims to produce a therapeutic effect through the manipulation of gene expression or through altering the biological properties of living cells.
This document provides an overview of gene expression systems and vectors used for gene transfer. It discusses the key phases of gene expression including transcription, post-transcriptional modifications, RNA transport, translation, and protein binding. It describes the two major categories of gene therapy - somatic and germline - and the three types of delivery - ex vivo, in situ, and in vitro. Finally, it summarizes the major viral vectors including retroviruses, adenoviruses, lentiviruses, and adeno-associated viruses, as well as various non-viral physical methods like electroporation and chemical methods using inorganic particles and biodegradable polymers.
The emerging CRISPR/Cas9 gene editing technology greatly accelerates the R&D process in life sciences. Here, we briefly introduce CRISPR/Cas9 and its delivery strategies.
The Recent advances in gene delivery using nanostructures and future prospectsAANBTJournal
This document summarizes recent advances in using nanostructures for gene delivery in gene therapy. It discusses the key challenges to effective gene therapy, including overcoming intracellular and extracellular barriers to delivery. It reviews the history of viral and non-viral gene delivery methods. Specifically, it describes several non-viral methods that have been developed using nanostructures, such as magnetic nanoparticles, PEGylated multi-component carriers, oligonucleotides, lipoplexes, polyplexes, and dendrimers. Overall, it finds that while viral methods remain more effective, recent advances in non-viral nanostructure-based delivery systems show promise for improving safety and effectiveness of gene therapy.
Gene therapy can be broadly defined as the transfer of genetic material to cure a disease or at least to improve the clinical status of a patient.
One of the basic concepts of gene therapy is to transform viruses into genetic shuttles, which will deliver the gene of interest into the target cells.
Safe methods have been devised to do this, using several viral and non-viral vectors.
In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patient's cells instead of using drugs or surgery.
The biggest hurdle faced by medical research in gene therapy is the availability of effective gene-carrying vectors that meet all of the following criteria:
Protection of transgene or genetic cargo from degradative action of systemic and endonucleases,
Delivery of genetic material to the target site, i.e., either cell cytoplasm or nucleus,
Low potential of triggering unwanted immune responses or genotoxicity,
Economical and feasible availability for patients .
Viruses are naturally evolved vehicles that efficiently transfer their genes into host cells.
Choice of viral vector is dependent on gene transfer efficiency, capacity to carry foreign genes, toxicity, stability, immune responses towards viral antigens and potential viral recombination.
There are a wide variety of vectors used to deliver DNA or oligo nucleotides into mammalian cells, either in vitro or in vivo.
The most common vector system based on retroviruses, adenoviruses, herpes simplex viruses, adeno associated viruses.
Gene Remedy: A New-Fangled Line of Attack to Pay for SicknessesBRNSSPublicationHubI
Gene therapy involves modifying genes to treat diseases. Genes contain the code for proteins that govern bodily functions. Mutated genes can cause diseases like cancer. Gene therapy aims to replace or suppress mutated genes. Genes are delivered using viral or non-viral vectors like viruses or plasmids. Ex vivo gene therapy extracts cells, inserts genes, and reinserts cells, while in vivo inserts genes directly. Clinical trials show gene therapy can treat diseases like hemophilia, leukemia, and blindness. However, safety issues around gene insertion and immune response remain challenges.
Gene therapy involves modifying genes to treat diseases. Genes contain codes to produce proteins that govern bodily functions. Mutated genes can cause diseases like cancer. Gene therapy aims to replace or suppress mutated genes using vectors to deliver new genes. Vectors include viruses modified to not cause disease and non-viral methods like naked DNA or liposomes. Gene therapy has shown promise in treating diseases like hemophilia, leukemia, and blindness, and is being studied for other untreatable diseases. However, safety issues remain to be addressed before it becomes more widely used clinically.
This document discusses adenoviral cloning vectors. It begins by defining a cloning vector as a small piece of DNA that can be stably maintained in an organism and have foreign DNA inserted into it for cloning purposes. It then discusses viral vectors, noting that they are commonly used to deliver genetic material into cells through transduction. The document focuses on properties of viral vectors, specifically safety features and targeting abilities. It provides details on adenoviruses, noting they can efficiently transfer genes, their structure, applications in gene therapy and vaccination, and their DNA genome capacity. Adeno-associated viruses are also mentioned as attractive for gene therapy due to mild immune response.
A good comprehensive review of gene delivery and gene therapy. especially for master of pharmacy 2nd-semester students as per the PCI syllabus of subject Molecular pharmaceutics.
List of contents under this ppt :
{A} GENE THERAPY
(1) Definition
(2) Introduction
(3) History
(4) Ex-Vivo gene therapy
(5) In-Vivo gene therapy
(6) Germline gene therapy
(7) Advantages of gene therapy
(8) Disadvantages of gene therapy
(9) Potential target diseases for gene therapy
a. inherited disorders :- ADA SCID, Chronic granulomatous, Hemophelia
b. Cancer
{B} GENE DELIVERY
(1) Definition
(2) Introduction
(3) Types of vectors
a. Viral :- Retrovirus, Adenovirus, Adeno associated virus, Herps simplex virus
b. Non viral :-
Physical methods - Gene gun, Microinjection, Electroporation, Sonoporation
Chemical methods - Oligonucleotides, Lipoplexes, Polyplexes, Dendrimers, Nanoparticles.
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RETROVIRUS MEDIATED GENE TRANSFER AND EXPRESSION CLONINGSrishtiRoy10
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The Production of Vaccines using Genetic Engineering as the world’s population continues to rise annually, new technology becomes known to man! Technology is a never-ending process where newer and better things are being discovered. The area of technology that will be discussed here is biotechnology. Biotechnology is the harnessing by man of the ability of organisms to produce drugs, food or other useful products. Micro-organisms are the main ones involved in biotechnology, especially bacteria and fungi. More recently, genetic engineering or the altering of the genes, the building blocks which determine the make-up of an organism, has been increasingly used in biotechnology.
1. 1
In silico vector engineering of recombinant
adeno-associated viral vectors for treatment of
Leber congenital amaurosis
Brett Davis
Senior Thesis
Endicott College Spring 2014
2. 2
Acknowledgments
I would like to acknowledge Dr. Jessica Kaufman for her assistance in completing this
project, along with Dr. Jason Nichol as my thesis advisor. I would also like to thank the
Biotech class of 2014 for their support.
3. 3
Table of Contents
Title Page Number
List of figures……………………………………………………………………………..4
Abstract with key words…………………………………………………………………..5
Introduction………………………………………………………………………………..6
Materials and Methods…………………………………………………………………...13
Results……………………………………………………………………………………19
Discussion………………………………………………………………………………..23
References………………………………………………………………………………..27
4. 4
List of Figures
Title Page Number
Figure 1: rAAV vector for LCA 2 (RPE65)……………………………………………..11
Figure 2: Genome Compiler main workshop area……………………………………….14
Figure 3: AAV inverted terminal repeat BLAST search results…………………………16
Figure 4: AAV vector template built in Genome Compiler……………………………..18
Figure 5: rAAV vector for LCA 1 (GUCY2D)………………………………………….20
Figure 6: rAAV vector for LCA 3 (SPATA7)…………………………………………...20
Figure 7: rAAV vector for LCA 4 (AIPL1)……………………………………………...20
Figure 8: rAAV vector for LCA 9 (NMNAT1)………………………………………….21
Figure 9: rAAV vector for LCA 11 (IMPDH1)………………………………………….21
Figure 10: rAAV vector for LCA 13 (RDH12)………………………………………….21
Figure 11: rAAV vector for LCA 15 (TULP1)…………………………………………..22
Figure 12: rAAV vector for LCA 16 (KCNJ13)…………………………………………22
5. 5
Abstract
The use of viruses as vehicles for the delivery of therapeutic DNA necessitates
engineering of recombinant vectors to be used in the treatment and testing of animal
models. It is necessary that the design process be done in silico to ensure efficiency and
plausibility of the gene therapy treatment. For the recombinant viral vector to be
engineered, it is not only necessary to incorporate therapeutic DNA, but also certain
features (e.g. promoter) needed for expression of the transgenic material. The goal of this
thesis project is to use Genome Compiler, a genetic design platform, to engineer adeno-
associated viral vectors for the treatment of different forms of LCA. The basis for the
design will be taken from Jacobson et al who successfully treated animal models with
knock-in LCA 2 (mutation in RPE65 gene) using recombinant AAV vectors. Genome
Compiler will allow for in silico vector engineering of many different types of LCA,
defined by mutations of different genes, using the published design of Jacobson et al as a
template.
Key Words: Adeno-associated virus (AAV), Leber congenital amaurosis (LCA), viral
vector design, in silico design, RPE65, Genome Compiler, synthetic design
6. 6
Introduction
Since its emergence on the biotech scene in the early 1990’s, the potential of gene
therapy has only been partially realized with roughly 1700 clinical trials since its
inception1. This period of time, while reflecting a certain level of progress and scientific
excitement, has been tumultuous and slow going1. The initial struggles in gene therapy
were primarily related to safety and biocompatibility concerns, which led to a significant
decrease in optimism that DNA based treatments would dominate the future of therapy1.
This adversity has more recently been replaced by success with positive results in treating
ADA deficiency, SCID-X1 and adrenoleukodystrophy, among others1. These cases have
built confidence and forward momentum for gene therapy studies in the current decade,
while diminishing the stigma towards such treatments from previous negative results. The
argument for utilizing gene therapy techniques has become more robust as the early
questions of safety and efficacy have been addressed.
The basic principle behind gene therapy, to use DNA as a therapeutic tool for
treating disease, is a simple one but complicated to implement and deliver 1, 5-7, 13, 14, 19.
The readily available and ever growing library of basic biological information (e.g.
sequence data) along with advances in genetic engineering processes has simplified the
design of DNA therapeutics 1,2. The difficulty arises in determining how a functional
copy of a gene can be delivered to a patient efficiently and safely. The primary form of
gene therapy involves packaging a functional gene into a vector to be delivered to the
treatment area. Vectors can be described as vehicles that carry foreign genetic material to
affected areas that can then be replicated and expressed for a desired, advantageous affect.
The type of vector determines the functionality; the therapeutic DNA copy is either
7. 7
incorporated into the host genome or delivered to the nucleus to be transcribed and
translated separately. These two results are most efficiently achieved by using viruses as
vehicles for delivering DNA to affected cells. The use of viruses in gene therapy has
resulted in the primary safety concern of biocompatibility in the process of delivering the
therapeutic DNA copy1, 3-7, 13-21. The aforementioned failure and subsequent successes in
gene therapy are directly related to an increased understanding of molecular medicine and
how viral vectors affect the body1.
In the recent history of gene therapy, retroviruses emerged as the vector candidate
that would improve the likelihood of success in clinical trials1, 5, 13-15. Retroviruses are
RNA based and utilize reverse transcriptase to become double stranded DNA from its
genome14. Inside of the nucleus, retroviruses incorporate into the host chromosome using
integrase, an enzyme produced by the virus that results in the provirus (incorporated in
host genome) state14. In the context of gene therapy, engineered genetic material (the
transgene) of another organism is incorporated into the RNA genome of the retrovirus.
The recombinant viral-human vector is delivered to affected cells that incorporate the
retroviral and transgenic DNA into the host genome. The desired result is the
transcription and translation of the therapeutic gene copy to make functional protein
capable of treating a certain disorder. The primary advantage of retroviral gene therapy is
that it allows for a possible permanent outcome and treatment. This is achievable due to
retroviruses being proviruses that will continue to be expressed after cell division,
resulting in more copies of functional transgenic protein being made 14, 15. The primary
concern for using retroviral vectors is, however, directly related to this genome
incorporating quality. This characteristic is inherently concerning in that it raises a
8. 8
distinct possibility of cancer causing oncogenesis and mutagenesis5, 13-15. Insertion of the
recombinant retroviral vector into host chromosomes is relatively random, and there can
be disruption of functioning genes or more critical genes controlling cell division5, 13-15.
In a recent study, the successful disease treatment of X-SCID using a retroviral gene
therapy approach had the adverse result of leukemia developing in several patients 5, 13-15.
This has caused gene therapy researchers to either develop solutions to address
oncogenesis of retroviral treatment or turn to other vector options 1, 5, 13-15, 24.
The primary response to retroviral vectors causing cancer was increased interest
in using adenoviral vectors 1, 24. Adenoviruses are comprised of a double stranded DNA
genome that enter cells but are transient; they do not incorporate into host cell
chromosomes. Contrary to retroviruses, adenoviruses have a desired affect as gene
therapy vectors by carrying transgenic DNA that is transcribed separately from the host
genomic DNA. Among the advantages of adenoviruses are the large size (37kb) for
incorporation of therapeutic DNA and the wide range of dividing and non-diving cell
types they can affect24. Additionally, the strains used to construct recombinant vectors are
well characterized and therefore have a predictable functionality24. The drawback in
using adenoviruses for gene therapy is the limited amount of time cells are able to
produce protein from the transgene. This is a result of the transient nature of
adenoviruses; failure to incorporate into the host genome addresses the issue of causing
cancer but expression fails to carry through cell division. Adenoviral treatments often
times require large amounts of product to have an impact on treating a certain disorder.
On the issue of safety, the high profile case in 1999 involving the death of a research
participant treated with an adenoviral vector was a clear message that adenoviruses can
9. 9
be particularly harmful to humans 25. This case limited the growth of adenoviral studies
greatly and in turn put gene therapy into a state of regress thereafter 1, 25.
The status of gene therapy as a treatment option was dependent on using viral
vectors that would be efficient, but would place the primary importance on safety. The
emergence of adeno-associated viral (AAV) treatments in the last seven years has
allowed for a reemergence and revival of gene therapy by addressing the principal issue
of safety 1, 3, 4, 16-21. The genome of the virus is 4.7 kb in length and is single stranded
DNA26. There are two open reading frames of the virus, described as rep (replication) and
cap (capsid). Rep is responsible for replication of the virus and cap encodes for the
protein envelope surrounding the DNA26. A key difference between AAV from
adenoviruses is the incorporation of the viral DNA into the host genome. Unlike
retroviruses, AAV have a designated site for integration on chromosome 9 in a 2 kb
region of the long arm26. The primary concern of gene therapy treatments being safety,
AAV has a distinct advantage in having no known pathogenicity 3, 6, 7, 19, 20. This non-
disease causing virus’ safety profile is supported by its limited immunogenicity or ability
to cause an immune response 6, 19, 20. Additionally, recent studies primarily associated
with retinal gene therapy have found that AAV has the ability to transduce post-mitotic
cells efficiently while maintaining high levels of expression 20. The primary drawback in
using AAV as a vector for gene therapy is its relatively small genome. AAV can only
package a certain amount of exogenous material; this includes an enhancer, a promoter, a
polyadenylation signal, and a human gene of interest. The amount of exogenous material
cannot surpass 4.7 kilobases, which limits the use of AAV in gene therapy for disorders
caused by mutations in larger genes.
10. 10
In 2006, the University of Florida did a proof of concept study using AAV vectors
to treat Leber congenital amaurosis 2 (LCA 2) in different animal models17. The positive
results of this study, improved vision and lack of immune response in at least 70% of
animals (for each experiment), paved the way for the 2009 study at University of
Pennsylvania in human subjects 4,17. This subsequent study, along with the safety and
efficacy update published in 2010, confirm successful treatment of LCA 2 using AAV
vectors 4, 16, 17.
Congenital disorders are genetic defects present from birth and in certain cases the
disorder’s symptoms can worsen throughout ones lifetime 8, 22. Leber congenital
amaurosis (LCA) is one such disorder, causing partial blindness at birth and worsening
sight later in life 4, 8, 22. More specifically, LCA describes a set of retinal dystrophies that
arise in early childhood with the primary symptoms of vision loss, nystagmus
(involuntary eye movement), and severe retinal dysfunction 8, 22. These symptoms occur
along with many others, such as photophobia (abnormal intolerance to light) and high
hyperopia (farsightedness), which contribute to an overall diagnosis of blindness 22. This
disorder affects an estimated 1 in 30,000 to 1 in 80,000 live births 22. LCA is primarily
identified through behavior of those afflicted in the first years of life 4, 22. In most cases
vision worsens and is lost entirely in a patient’s 30’s or 40’s 4. LCA has historically been
viewed as a managed disease rather than a treatable one, with correction of refractive
error and use of low vision aids being the primary sources of managing the disorder while
patients still retain some vision22. There are currently sixteen types of LCA defined by
OMIM, with mutations in specific genes accounting for each variation of the disorder 8.
11. 11
This thesis project involved the use of bioinformatics tools to design novel adeno-
associated viral vectors (AAV) for treating different forms of Leber congenital amaurosis
(LCA). This was be accomplished by modeling the vector designs after those used in the
initial proof of concept studies (2006) that used adeno-associated viral vectors to treat
LCA 2. The image below describes the features of the two rAAV used in the POC study
(Figure 1)17. The human cytomegalovirus (CMV) immediate early (ie) enhancer is
present to ensure cell-type-specific gene expression 27. The inverted terminal repeats on
both ends of the vectors are structures of AAV that are 145 base pairs long and are
necessary for replication of the genome 26. Simian virus (SV) 40 polyadenylation
(poly(A)) signal is an RNA sequence that adds poly(A) tails to mRNA sequences to allow
for maturation28. This process is essential for eventual translation and expression of the
transgenic material of the recombinant viral vector28.
Figure 1: (A) AAV2-CBo-hRPE65, 4070 bp, and (B) AAV2-CBSB-hRPE65, 3921 bp.
The two vectors differ by 152 bp at the 5' end of the CMV immediate early enhancer
(long vs. normal). ITR describes the AAV2 inverted terminal repeat, followed by the
CMV immediate early enhancer. This is preceded by chicken B-actin (CBA) promoter,
Exon 1, intron, and Exon 2. The transgene, human RPE65 cDNA, follows Exon 2. The
12. 12
design is concluded by the presence of SV40 polyadenylation signal before the opposite
ITR. 17
The growing field of gene therapy along with increased advances in molecular
biology and medicine has signaled a need for in silico design of viral vectors to be used
in clinical trials. In silico, or ‘performed by computer’, is a necessary step for vector
design that occurs before ex vivo and in vivo studies of affects on animal models or
human tissue/subjects can take place 2. The growing field of bioinformatics, management
information systems for molecular biology, has made this possible on two levels 2. Firstly,
the growth of basic biological information databases has supplied researchers and
programmers alike with readily available and accessible sequence data 2. Secondly,
programs and tools are now being developed with specific goals in mind (e.g. building
phylogenic trees showing relationships between organisms) that make use of the
exponentially growing databases 2. Vector design in silico is now possible with the
development of programs that use sequence information input to design novel,
recombinant viral vectors. Genome Compiler is one such program and was used in this
thesis project to design AAV vectors with aforementioned features for the treatment of
different forms of LCA.
13. 13
Materials and Methods
The three vector design programs that were chosen to be tested were Benchling,
Genome Compiler, and Gene Designer. It was necessary to assess the program’s relative
strengths and weaknesses on the following criteria: interface, usability, robustness,
aesthetics of design, and workflow. This evaluation ensured the best program was chosen
to design quality vector(s) with relative ease. To accomplish this, test vectors were
assembled in silico to be used as benchmarks for the robustness and aesthetics of design.
The act of assembly itself was the inherent measure for interface, usability, and
workflow. After three recombinant adeno-associated viral vectors were assembled, it was
found that Genome Compiler had strengths in all the criteria desired, most notably in
usability and interface. It was therefore chosen as the program to be used in designing
vectors for the thesis project.
The Genome Compiler program was installed in conjunction with Adobe Air on a
MacBook Pro laptop running OS X Mountain Lion. Genome Compiler makes use of a
workshop style interface that was used to construct and manipulate vectors from DNA
sequences. The National Center for Biotechnology Information (NCBI) search tool
incorporated in the program was used to import desired sequence information. In cases
were a certain sequence or element could not be found easily the genInfo identifier (GI)
number was copied from the NCBI page directly and searched for in Genome Compiler.
After importing or uploading from NCBI the program sorts the sequences by type (e.g.
Viral DNA) into folder in the library under Materials. It is ideal that DNA sequences be
found using NCBI search; if this search only results in mRNA sequence data, it can be
14. 14
‘translated’ to a DNA sequence automatically in the program. Genome Compiler utilizes
a drag-and-drop workflow that allows the user to easily build on desired templates.
Figure 2: Genome Compiler main workshop area. NCBI search tool, library sorting, and
drag and drop features shown. Projects are built in the blank area by incorporating
desired elements into imported templates.
The initial strategy used to build the AAV vector template (viral DNA and certain
features used by Jacobson et al) started with the complete viral genotype of AAV
serotype 2. The inverted terminal repeats (ITRs) of AAV could then be located in the
DNA sequence, labeled, and used as markers to build between in Genome Compiler.
Human genes that responded to different LCA types could then be inserted using the final
template. The exclusion of viral DNA outside the ITRs, however, led to a revised strategy
of importing sequences independently and piecing them together in Genome Compiler.
The final result would be the inverted terminal repeat of adeno-associated virus serotype
2 followed by cytomegalovirus (CMV) immediate early (ie) enhancer, chicken beta actin
(CBA) promoter, simian virus (SV) 40 polyadenylation (poly(A)) signal, and the reverse
complement of the ITR sequence. This template could then be used to insert human DNA
in silico for each LCA type between the promoter and poly(A) signal.
15. 15
The ITR of AAV-2 nucleotide sequence was searched for in NCBI outside of
Genome Compiler. This method proved to be more useful; the title of the nucleotide file
often times did not specifically describe the relevance of the sequence. The ITR sequence
used was a synthetic construct under a certain patent number (Sequence 1 from Patent
WO0192551); it was therefore necessary to import this sequence rather than attempting
to locate it by name alone in Genome Compiler. The accession number for this file was
used to run a standard nucleotide BLAST, or basic local alignment search tool. This
patent sequence for the ITR of AAV-2 was searched against the ‘others’ database (non-
human and non-mouse sequences) optimizing for highly similar sequences (megablast).
The BLAST compared the nucleotide sequence of the ITR synthetic construct against
viral DNA sequences in the database and reported query covers for highly similar
sequences. It was found that in comparison to ‘Adeno-associated virus 2 right terminal
sequence’ there was a 100% match in identities as seen in Figure 3 below. This did not
confirm, however, that the synthetic ITR sequence was of the correct length. The known
length of 145 bases did not correspond to the 198 bases of the synthetic ITR sequence.
This determined that the nucleotide sequence needed to be truncated by 53 bases after
importation to Genome Compiler. A simple deletion of these bases was performed and
the sequence was labeled ‘Inverted terminal repeat of AAV serotype 2’.
16. 16
Figure 3: BLAST result comparing query to ‘Adeno-associated virus 2 right terminal
sequence’. There was a 145/145 (100%) match between the two sequences suggesting the
nucleotide sequence is highly relevant and can be imported into Genome Compiler
The next sequence to be imported into the program was the CMV ie enhancer
sequence. The NCBI search resulted in synthetic constructs under patent numbers much
like the search for the ITR sequence; the same strategy and approach was implemented in
importing the sequence to Genome Compiler. A BLAST search was used aligning the
selected nucleotide file for a CMV ie synthetic construct against the ‘others’ database for
highly similar sequences (megablast). The results of this search needed to be more
thoroughly investigated than those of the ITR search; enhancer regions can often be
modified and/or combined with different promoters as a single synthetic construct. Three
different CMV enhancer synthetic construct sequences of varying lengths were used as
queries against the others database. The compiling of the information (query cover,
statistical significance, and identity matches) confirmed the most relevant sequence to
import to Genome Compiler. It was dragged into the workspace immediately following
the AAV-2 ITR sequence.
17. 17
The inclusion of the chicken beta actin (CBA) promoter involved the same
process as importing the enhancer sequence. BLAST searches confirmed the relevancy of
the CBA promoter sequence found in NCBI’s nucleotide database. To further confirm the
results, a program called SnapGene, a bioinformatics tool for designing PCR primers,
was used to import the available CBA sequence in the programs’ database to Genome
Compiler for comparative purposes. The comparison showed no differences in the CBA
sequences imported from NCBI and SnapGene’s databases. The sequence was labeled in
Genome Compiler and placed immediately following this CMV ie enhancer sequence. As
previously mentioned, enhancers and promoters are often grouped/built together in
synthetic biology. The CMV ie enhancer / CBA promoter combination is not an
uncommon one and has been used in many studies for viral vector construction. To
further verify sequence relevance, the combined nucleotide sequence of the CMV ie
enhancer and CBA promoter was searched against the others database for highly relevant
sequences in BLAST. The results of this search confirmed that the same nucleotide
sequence appears in many other sequences of the database that respond to synthetic
constructs used for viral vectors or other purposes.
The final sequence that needed to be imported to Genome Compiler was the
simian virus (SV) 40 poly(A) signal. The NCBI search resulted in a nucleotide file of the
complete genome of SV 40. This complete genome file contained certain keywords that
differentiated the sections of the genome. It was not clear that a certain nucleotide
sequence responded to the poly(A) signal, prompting the need for further investigation.
The potentially correct sequence was copied to PolyApred, a program that predicts
polyadeylation signal sequence relevance based on signature sequences scores. Poly(A)
18. 18
signals have signature sequences (e.g. AATAAA) that can simply be searched for by the
program and matched. The positive match in PolyApred was confirmed by a subsequent
BLAST search of the sequence against the others database for highly relevant sequences.
The SV40 poly(A) signal sequence was imported into Genome Compiler and placed
immediately following the CBA promoter.
To complete the AAV-2 vector template, the final step was taken to add the ITR
to the opposite of the sequence. In Genome Compiler the reverse complement of the
previously imported ITR sequence was copied and placed immediately following the
SV40 poly(A) signal. This action completed the template that was then used to build
recombinant vectors for treating different forms of LCA with human DNA that responded
to each type. Figure 4 shows the final template along with an inserted ‘gap’ section
between the promoter and poly(A) signal to be replaced by human DNA.
Figure 4: Inverted terminal repeat (ITR) of AAV serotype 2 (145 bp), Cytomegalovirus
immediate early (CMV ie) enhancer (427 bp), Chicken Beta Actin (CBA) promoter (278
bp), gap (gene of interest for LCA type), Simian virus 40 polyadelynation signal (SV 40
poly(A)) signal (240bp), reverse complement of inverted terminal repeat (ITR) of AAV
serotype 2 (145 bp). 945 bp of exogenous material is present.
The final step involved importing human genes that correspond to different forms
of LCA; sixteen forms of LCA are listed by OMIM. LCA 2 and the RPE65 gene was
excluded as this was the form of LCA and gene targeted by Jacobson et al, whose vector
designs were used as a model in this study. Other forms of LCA were excluded due to
19. 19
size of the gene; previous studies and research has suggested genes that are relatively
large are incompatible with the small AAV genome. The threshold of 4.7 kb of
exogenous material was established29. The enhancer, promoter, and poly(A) signal
together were 945 bp in length, leaving 3.755 kb for human DNA to be incorporated in
the final designs. This size limitation included the following types of LCA and their
respective genes: LCA1 (GUCY2D), LCA3 (SPATA7), LCA4 (AIPL1), LCA9
(NMNAT1), LCA11 (IMPDH1), LCA13 (RDH12), LCA15 (TULP1), and LCA16
(KCNJ13). NCBI searches were utilized to import the human genes into Genome
Compiler corresponding to the different forms of LCA. The nucleotide sequences were
then incorporated into the AAV-2 vector template between the promoter and poly(A)
sequences. The results were eight AAV-2 vectors built in silico for treating different
forms of LCA.
20. 20
Results
Figure 5: Inverted terminal repeat (ITR) of AAV serotype 2 (145 bp), Cytomegalovirus
immediate early (CMV ie) enhancer (427 bp), Chicken Beta Actin (CBA) promoter (278
bp), Homo sapiens retinal guanylyl cyclase 1 (GUCY2D) gene (3.641 kb), Simian virus
40 polyadelynation signal (SV 40 poly(A)) signal (240bp), reverse complement of
inverted terminal repeat (ITR) of AAV serotype 2 (145 bp). 4.586 kb of exogenous
material is present. GUCY2D is the gene that defines LCA 1.
Figure 6: Inverted terminal repeat (ITR) of AAV serotype 2 (145 bp), Cytomegalovirus
immediate early (CMV ie) enhancer (427 bp), Chicken Beta Actin (CBA) promoter (278
bp), Homo sapiens spermatogenesis-associated 7 (SPATA7) gene (1.935 kb), Simian
virus 40 polyadelynation signal (SV 40 poly(A)) signal (240bp), reverse complement of
inverted terminal repeat (ITR) of AAV serotype 2 (145 bp). 2.880 kb of exogenous
material is present. SPATA7 is the gene that defines LCA 3.
Figure 7: Inverted terminal repeat (ITR) of AAV serotype 2 (145 bp), Cytomegalovirus
immediate early (CMV ie) enhancer (427 bp), Chicken Beta Actin (CBA) promoter (278
bp), Homo sapiens aryl-hydrocarbon interacting protein-like 1 (AIPL1) gene (2.247 kb),
Simian virus 40 polyadelynation signal (SV 40 poly(A)) signal (240bp), reverse
complement of inverted terminal repeat (ITR) of AAV serotype 2 (145 bp). 3.192 kb of
exogenous material is present. AIPL1 is the gene that defines LCA 4.
21. 21
Figure 8: Inverted terminal repeat (ITR) of AAV serotype 2 (145 bp), Cytomegalovirus
immediate early (CMV ie) enhancer (427 bp), Chicken Beta Actin (CBA) promoter (278
bp), Homo sapiens nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) gene
(3.781 kb), Simian virus 40 polyadelynation signal (SV 40 poly(A)) signal (240bp),
reverse complement of inverted terminal repeat (ITR) of AAV serotype 2 (145 bp). 4.726
kb of exogenous material is present. NMNAT1 is the gene that defines LCA 9.
Figure 9: Inverted terminal repeat (ITR) of AAV serotype 2 (145 bp), Cytomegalovirus
immediate early (CMV ie) enhancer (427 bp), Chicken Beta Actin (CBA) promoter (278
bp), Homo sapiens IMP (inosine 5'-monophosphate) dehydrogenase 1 (IMPDH1) gene
(2.526 kb), Simian virus 40 polyadelynation signal (SV 40 poly(A)) signal (240bp),
reverse complement of inverted terminal repeat (ITR) of AAV serotype 2 (145 bp). 3.471
kb of exogenous material is present. IMPDH1 is the gene that defines LCA 11.
Figure 10: Inverted terminal repeat (ITR) of AAV serotype 2 (145 bp), Cytomegalovirus
immediate early (CMV ie) enhancer (427 bp), Chicken Beta Actin (CBA) promoter (278
bp), Homo sapiens retinol dehydrogenase 12 (RDH 12) gene (1.934 kb), Simian virus 40
polyadelynation signal (SV 40 poly(A)) signal (240bp), reverse complement of inverted
terminal repeat (ITR) of AAV serotype 2 (145 bp). 2.879 kb of exogenous material is
present. RDH12 is the gene that defines LCA 13.
22. 22
Figure 11: Inverted terminal repeat (ITR) of AAV serotype 2 (145 bp), Cytomegalovirus
immediate early (CMV ie) enhancer (427 bp), Chicken Beta Actin (CBA) promoter (278
bp), Homo sapiens tubby-related protein 1 (TULP1) gene (1.980 kb), Simian virus 40
polyadelynation signal (SV 40 poly(A)) signal (240bp), reverse complement of inverted
terminal repeat (ITR) of AAV serotype 2 (145 bp). 2.925 kb of exogenous material is
present. TULP1 is the gene that defines LCA 15.
Figure 12: Inverted terminal repeat (ITR) of AAV serotype 2 (145 bp), Cytomegalovirus
immediate early (CMV ie) enhancer (427 bp), Chicken Beta Actin (CBA) promoter (278
bp), Homo sapiens potassium inwardly-rectifying channel, subfamily J, member 13
(KCNJ13) gene (3.376 kb), Simian virus 40 polyadelynation signal (SV 40 poly(A))
signal (240bp), reverse complement of inverted terminal repeat (ITR) of AAV serotype 2
(145 bp). 4.321 kb of exogenous material is present. KCNJ13 is the gene that defines
LCA 16.
23. 23
Discussion
Of the sixteen different types of Leber congenital amaurosis, eight adeno-
associated viral vectors were designed. The LCA types excluded due to the payload
capacity of AAV were all within 1.2 kb of permitted exogenous material with the
exception of LCA 10 (CEP290 gene). The 4.7 kb limit for inclusion of foreign DNA is
not definitive; while the use of larger genes for AAV gene therapy have lacked efficacy,
this does not suggest AAV should be abandoned for slightly larger gene sizes.
Additionally, the relatively large sizes of the CBA promoter and CMV ie enhancer limit
the amount of human DNA to be incorporated and could be substituted to increase gene
capacity in AAV. Genome Compiler could be used to design the following LCA types
beyond the set payload limitation of 4.7 kb: LCA 5 (LCA 5 gene), LCA 6 (RPGRIP1),
LCA 7 (CRX), LCA 8 (CRB1), LCA 12 (RD3), and LCA 14 (LRA). The genes that
correspond to these types of LCA range from 3.95 kb to 4.909 kb. The CMV ie enhancer,
CBA promoter, and SV 40 poly(A) signal present in the design vectors was 945 bp in
total length. The use of shorter sequences could bring this total down to approximately
500 bp. This step alone would allow for packaging of RPGRIP1, CRB1, and RD3 to treat
the respective LCA forms of LCA 6, LCA 8, and LCA 12. The remaining three forms
(LCA 5, LCA 7, and LCA 14) could be designed and used as upper limitation testers for
AAV in a laboratory setting.
The genetic heterogeneity of LCA describes 16 known genes associated with
retinal dystrophies. The respective percentages of each LCA type in the population are
currently unknown; it is unclear whether or not the designed AAV vectors for eight
different types of LCA represents a great percentage of the population. It is estimated that
24. 24
LCA 10 (CEP290 gene) accounts for approximately 21% of all LCA cases. CEP290 is a
relatively large gene (7.97 kb) and well beyond the small payload capacity of AAV. LCA
10 is the sole form of LCA that would have predictably low success if a gene therapy
approach was taken using AAV as a viral vector packaged with human CEP290.
Adenoviruses and retroviruses have much larger exogenous packing capacity due to their
sizes. CEP290 could be packaged in a viral vector of one of these two categories and
designed in silico using Genome Compiler. As mentioned previously, there is an inherent
concern of safety (pathogenicity and immunogenicity) associated with these types of
viruses and their usage in gene therapy treatments. The concerns of safety and efficacy of
a proposed adenoviral or retroviral design could be addressed in animal model studies.
The sheltered treatment area of the eye and retina suggest limited adverse affects in using
these types of viruses. Additionally, the efficacy of such a study could be weighed against
using AAV to treat LCA types with genes above the 4.7 kb packaging capacity. This
would require additional animal model testing with an adenoviral or retroviral approach
for delivery of human LCA 5 gene, CRX gene, and LRAT gene responding to LCA types
5, 7, and 14 respectively. These vectors could be designed in silico using Genome
Compiler.
The primary future consideration for designing AAV vectors for different types of
LCA is to order the designs to be made synthetically. This process can be carried out in
Genome Compiler by ordering designs to be constructed and delivered as gene therapy
treatments. The study performed by Jacobson et al could be used for the basis of animal
model proof of concept research. The results of this study suggested high efficacy and
safety that led to human clinical trials for treatment of LCA 2 (RPE65 gene). These
25. 25
results could act as a benchmark for the following qualities: initial visual improvement,
retinal function, and duration of improved vision after treatment. Visual improvement can
be measured using standard procedures for proper function testing in dogs, the ideal
model for testing gene therapy treatment for LCA. Retinal and ocular improvement can
be viewed visually with histological comparison and staining. Safety could be measured
using standards for biodistrubution and immunogenicity related to dosage level. The
amount of AAV vector to be administered may vary for each LCA type, despite the
design being identical outside the human DNA incorporated. The results of a collective
study would confirm the validity of designed AAV vectors for each LCA type. The
potential success of such a study would pave the way for human clinical trials for those
afflicted with a wide variety of gene mutations related to different types of LCA.
The design program utilized in this thesis project, Genome Compiler, had great
strengths in interface and ability to simply import and organize relevant sequences. The
program could, however, be improved and optimized for gene therapy viral vector
design. As gene therapy becomes more relevant in molecular medicine, there is a need to
improve in silico design to expedite the process to eventual synthetic construction of
therapeutics. Genome Compiler could implement viral templates including features such
as inverted terminal repeats (ITRs) to manipulate in the process of gene therapy design.
Common features such as the chicken beta actin (CBA) promoter could be loaded into the
program and available for simple selection and incorporation into designs. NCBI search
within Genome Compiler is often difficult due to the lacking function of opening
sequence files. Synthetic construct sequences are often listed under patent numbers and
not the relevant name; it is necessary to using the NCBI website for further investigation
26. 26
prior to importing the sequence. The addition of an in-house BLAST tool would allow
users to analyze the relevancy of sequences by identity matching before incorporation
into vector designs. These small changes would improve Genome Compiler as a gene
therapy design tool and potential improve the robustness and viability of a potentially
constructed viral vector.
27. 27
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