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Gene Therapy Essay Topics

Essay on Gene Therapy!

Essay on the History of Gene Therapy:

Gene therapy was conceived in 1960, the breakthrough was the synthesis of recombinant DNA molecule (rDNA) in 1972. The rDNA molecules were first duplicated and grown in bacteria in 1973. Later split gene was discovered through the use of recombinant DNA technologies in 1977. Two independent techniques were developed in 1977 to determine the DNA sequences that contain genetic information.

This facilitated direct inspection of the genetic material and analysis of its functions. A strain of Pseudomonas bacterium was developed later in 1980 that could digest certain petrochemical. These techniques lead to the launch of Humulin i.e. human insulin in 1982. Recombinant DNA techniques were first used for the prenatal detection of sickle cell disease in 1982.

The first inherited alteration of genes in the germ line of mice was achieved in 1980, when the gene for rat growth hormone was introduced into mice/this lead the growth of mice to double in comparison to usual size. Zinc in diet was used as regulator/inducer which maneuvered rat growth hormone inserted genes.

The interaction between zinc and growth hormone gene was due to special DNA sequence (in the growth hormone gene).The progeny of the genetically altered mice inherited the new gene, to make them grow twice in size.

A. Genes and Expression of Traits:

Single gene associated mutation leads to genetic diseases. This would be corrected by single gene therapy, which is relatively easy as compared to multiple gene based traits or its interaction with environment. Such diseases are called single gene defects. Most diseases have a mixture of genetic and environmental contributions. In disorders like Huntington or Tay-Sachs diseases, the influence of a single gene is so vast that the disorders are called genetic diseases.

(a) Mendelian Traits:

The diseases that are primarily due to a single gene defect/deficiency follow the Mendelian laws of inheritance. The similar patterns of inheritance have been observed in various human diseases, and thus indicate gene associated diseases. The disease due to molecular defects in non-enzyme proteins has also been observed.

It is sickle cell anemia, in which an abnormal hemoglobin protein has been found. Many genetic diseases are due to changes in just a single gene, such as Adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) enzyme deficiencies among human.

Disorders such as sickle cell anemia, familial hypercholesterolemia, polycystic kidney disease, Huntington disease, neurofibromatosis, Duchenne muscular dystrophy, cystic fibrosis, achondroplasia, hemophilia etc. are due to single gene disorders.

The prevalence of disease like Alzheimer and hemochromatosis has been influenced by genetics. Single gene traits vary in expression, depending on its interaction with other genes and environment. The degree to which individual subject manifests signs and a symptom of genetic disease is termed as expressivity. Diseases can also be expressed with different intensity in populations, affecting some people and not others even though carrying the concerned gene.

This is called penetrance. If all having the defective gene also express the disease is called complete penetrance, while incomplete penetrance is when some people have the gene but not the disease. Single gene traits/ disorders can be recessive, dominant, or X-linked.

(b) Recessive Disorders:

When one receives faulty gene from both parents is termed as recessive disorder. Disorders due to dysfunctional gene pairs are mostly due to protein abnormalities that lead to a biochemical imbalance. Sickle cell anemia and thalassemia affect the protein part (globin) of hemoglobin, which carries oxygen via the blood to tissues.

Other recessive disorders, such as Tay-Sachs disease, ADA and PNP deficiencies, and phenyl ketonuria (PKU) affect enzymes whose absence or dysfunction adversely affects cellular metabolism. Most of the relatively well understood genetic diseases are recessive disorders that can be linked to specific defects in enzymes.

(c) Dominant Disorders:

In most dominant disorders the biochemical nature of the abnormality has not been observed. The molecular defect in dominant disorders is not established as it is for recessive ones. When offspring having just one defective gene (from one of its parent), lead to expression of the disease is called dominant disorder. This include abnormal disruption of biochemical pathways followed by heme degradation due to deficiency of enzymes.

(c) .1. X-Linked Disorders:

The abnormalities which are carried through X chromosome are termed as X- linked diseases. In general males are affected as males have only one copy of the X chromosome. The inheritance pattern of X-linked disorders is distinctive: male gets the traits only from mother side and “Y” from father.

Female can get a defective gene from either parent, but do not usually have the disease unless they get the abnormal gene from both the parents. X-linked traits thus act like dominant traits inherited only from the mother in male, and are usually recessive in female. X-linked disease traits in human are hemophilia, Duchenne muscular dystrophy, and Lesch-Nyhan syndrome.

There are apparently few traits, and no known diseases, that are carried in genes located on the Y chromosome, and expressed only in males. Single gene defects are, in general, the best understood of genetic diseases; the early instances of gene therapy might well be done to correct the effects of single mutant genes.

Multi-gene traits are certain traits that build up from the interactions of numerous genes democratically and therefore do not comply with simple Mendelian inheritance. Eye and hair color are the traits that are specified by such pattern. There are certain diseases as a result of interactions of multiple genes that are least affected by environmental influences.

(d) Environmentally Modified Traits:

Many traits in individuals are determined phenotypically by an interaction of genetic predisposition with the environment. There are diseases derived from interactions of genes and the environment in which both components contribute to the disease progression.

Diabetes mellitus in human at younger age is supposedly caused by a special susceptibility of insulin secreting cells to certain viral infections or other environmental exposure. There is little doubt that genetics and environment interact, but there is serious conflict whether which factor predominates.

B. Genetic Correction:

Gene therapy has been observed in animals and lower organisms. Genetic correction has been attempted successfully in fruit flies and mice. New genes have been incorporated in mammalian cells, but the gene inserted into chromosomal or cellular locations is yet to be predicted or regulated for their action. Genes transfer has been attempted to progeny in mice i.e. the gene for rabbit hemoglobin and rat growth hormone.

The growth hormone experiment was important as it had expression of the gene under manipulation, and inherited to the progeny of the treated mice. Although genetic manipulations have occasionally leaded to undesired effects i.e. sterility or induction of new mutations. Animal experimentations have leaded to change in germ line mostly therefore recent attempt is being based on somatic cells of animals.

These are more analogous to early human trials. Gene has been inserted through modified virus vector into bone marrow cells of mice and created proteins successfully. Similar mode may be used in animals/humans as well.

However apprehension of reversion of infection among viruses, induction of fresh mutations, cancer and integration into the germ line may appear as the study would proceed. Proper regulation of gene expression in the cells of humans and other higher animals is more intricate as compared to fruit flies and bacteria.

A. Feasibility of Gene Therapy in certain Genetic Disease:

(a) 1. Chromosomal Disorders:

In contrast to genetic diseases of single genes or few genes, there are few disorders caused by abnormal chromosomes. There are group of genetic disorders due to surplus or shortage of chromosomes. Subjects may have an abnormal number of chromosomes or its parts i.e. Down syndrome. Correction of such chromosomal anomalies is practically not possible by gene therapy as chromosomes typically contain numerous genes.

(a) 2. Dominant Traits:

Few dominant traits might be treated by gene therapy i.e. enzyme defects. These are inherited as dominant traits. There are diseases caused by deletions of small amounts of DNA and that could be replaced i.e. in retinoblastoma and Wilm’s tumor (cancers in childhood that are inherited as dominant traits).

Few dominant disorders have been characterized biochemically, and simple gene insertion may not correct many dominant disorders. Correction of dominant diseases may require insertion of huge amounts of DNA and/or gene surgery to remove the defective gene. Techniques for these complex manipulations have not been demonstrated in mammals. Therefore such therapy of dominant disorders is supposedly will take long to appear.

B. Criteria for Gene Therapy:

It should be based on scrutiny of risks and benefits for the individual patient with the thought of its wider implications. The factors considered should include latent efficacy, safety, dependability, alternative treatments if any, gravity of symptoms, and prognosis. This would be needed to be customized for particular genetic disease except some common factors which is applicable to the technique of gene therapy as such.

(b) 1. Safety, Efficacy and Reliability:

It would be necessary to judge safety and efficacy on animal data, procured in experiments that involve gene transfer. However, it would be tough to ascertain clinical benefit in the animals. Experiments might be performed, using the same gene and delivery system as would be used in human and the animals observed to see if they express the gene or develop side effects. Safety includes not only short term effects, but also long term consequences that may require years to confirm.

The effects in the next generation would be tough to assess especially if it involves germ line cells. The germ line effect has been observed in chemotherapy and radiation therapy involving tumour. There is risk of inducing fresh mutations in the subject which can ultimately be through to next generation. If experiments involve gene therapy to the patient, then impact over germ line may be a matter of concern.

It would require to see the risk benefit ratio. There are some specific concerns of using viruses as a vector to transfer DNA. There is possibility of its attaining virulence owing to reshuffling of genetic material in the host. So it would be prerequisite to design DNA not let the virus revert to being infectious.

It would be required to regulate the insertion and integration of DNA into that of the host cell, which may lead to fresh mutations in the cells. It may rarely lead to develop neoplasm. Determination of safety would be supposed to be derived from experiments on animals.

Among human, gene therapy has been considered depending upon severity of diseases, which included deficiency like ADA and PNP, urea cycle defeats, or Lesch-Nyhan syndrome. In these cases there is a complete lack of therapy, therefore treatment may have risk. This may further pave the way if remained safe. However, it needs to be repeatable and reliable as well.

(b) 2. Techniques of Gene Therapy:

Molecular genetics is evolving at a brisk pace from its infancy. It emerged after the discovery of DNA replication. DNA has been subjected to alteration during early seventies by specific cutting and reassembly further. The modified DNA was later reintroduced into cells. Regulation of the genes expression needs to ensure that the inserted gene (in DNA) will have desirable functions in the concerned cells and would not act otherwise.

Genes are copied and passed on by DNA replication. Genetic information is transmitted from one cell to its progeny by duplication or replication of its DNA. When a cell divides, it copies its DNA and distributes a copy to each of two offspring cells. A new therapeutic gene introduced into a cell in the laboratory can thus be reproduced through the process of cell division when the cell is placed into a patient and proliferates.

(b) 3. Isolation and Cloning:

In the first place of gene therapy it needs precise location of the disease causing gene. Following identification, the corresponding normal gene needs to be isolated. Later the normal single gene is being identified, isolated and cloned. This cloning includes combining the gene of interest with DNA sequences that facilitate/allow it to be copied, later placed in bacteria, yeasts or mammalian cells grown in culture.

This facilitates copying of DNA with the proliferation of cells. Further huge number of copies of DNA is purified. Later the gene of interest is spliced away from undesirable DNA sequences. This leads to millions or billions of copies of a single gene. These copies are then integrated with the DNA suitable for insertion into cells in vivo.

C. Types of Gene Therapy:

Different mechanisms of gene therapy:

Gene therapy refers to the insertion of genetic material to correct a defect.

This may be accomplished by:

(a) Introduction of a new version of gene into a cell, termed as gene insertion.

(b) Alteration of already placed gene in cell is termed as gene modification, and

(c) Excision and or replacement (of normal counterpart) of a particular gene in cell are called gene surgery.

Such genetic alterations would involve insertion of new material that directly codes for proteins or that affects how existing genes are expressed by suppressing or enhancing production of particular proteins.

Approaches for gene modification:

(a) Gene replacement:

Mutant gene sequence from host genome is removed and replaced with the normal functional host gene.

(b) Gene correction:

The mutant segment of gene is modified to let the gene function normally, without changing the gene into its natural form.

(c) Gene augmentation:

The expression of mutant gene in abnormal cell is modified by introduction of a normal gene sequence into the host genome. The defective host gene remains unaltered.

Transfer of Gene/s: Ideal System:

The ideal system to deliver DNA should

(i) Accommodate a broad size range of inserted DNA

(ii) Be available in a concentrated form

(iii) Is easily produced,

(iv) Target to specific types of cells

(v) not permit replication of the DNA

(vi) Provide long term gene expression and

(vii) Be nontoxic and non-immunogenic.

Introduction of genes in cells:

The method of introduction of gene/s can be:

(i) Biological method:

Viral vectors, mammalian artificial chromosome etc.

(ii) Chemical method:

Liposomes, ligand mediated gene delivery.

(iii) Physical method:

Microinjection, electroporation, gene gun, direct parenteral injection

The most common and relatively feasible method is the biological method. Viruses have a simple structure containing a protein coat and nuclear material. They contain sequences in RNA/DNA that permit insertion of genes in to the host DNA. This makes viruses ideal for gene delivery. Various viruses are used for these purposes such as retrovirus, adenovirus, vaccinia and herpes simplex virus.

The gene responsible for viral replication is replaced by replication incapable foreign genes using recombinant DNA techniques among retro viruses. This yields a generation of retroviruses lacking the structural genes required for replication.

A packaging cell line is engineered by transfecting:

(i) The gag gene which corresponds to group specific antigens,

(ii) The pol gene responsible for reverse transcriptase and

(iii) The integrase and env gene required for the envelope protein.

D. Delivery of Gene into Cells:

A ‘delivery vehicle’ called a vector is used to get the therapeutic material into the patient’s target cells.

The DNA with the normal gene can be administered to cells in vivo in various ways like :

(i) Using viruses,

(ii) Physically injecting it,

(iii) Treating the DNA chemically so that cells take it up,

(iv) Treating the cells so that they are induced to take in the DNA, or

(vi) By fusing the cells with membranes that contain the DNA. In the distant future, designed viruses or genetic elements may be used to transfer genes to specifically targeted cells in vivo.

(d) 1. Viruses:

Viruses usually contain genetic material encapsulated with an external coat of protein or membrane. It is due to their being relatively simple, controllable, and contains sequences that permit insertion of genes into the host’s DNA. This feature has made it an apt target to be exploited for gene transfer. Designed/modified viruses may be good candidate for gene therapy in the time to come.

It is due to:

(i) Their being highly efficient,

(ii) Can affect many cells, and are

(iii) Relatively easy to manipulate in the laboratory.

Attempts are being made in developing viruses that would

(i) Not injure cells,

(ii) Not propagate uncontrollably, and

(iii) Enter only target cells.

Such model viruses have been used with success to insert foreign genes into haemopoietic cells in mice. Viruses are highly evolved natural vectors for the transfer of foreign genetic information into cells.

This characteristic of viruses has led many attempts to engineer recombinant viral vectors for the delivery of therapeutic genes into diseased tissues. However the gene therapy is at the stage of infancy which needs refinement to cater such vector before letting gene therapy become standard care for any individual disorder.

(d) 2. Viral vectors for gene therapy:

Viruses have properties to introduce their genes to specific animal cells as a mode of their multiplication (as observed with the specific tissue they affect). These vectors can be customized and made as per the interest by substituting viral genes with therapeutic genes. Some gene therapy trials involving viral vectors has been approved in UK. Few modified viral vectors include pox viruses, adenoviruses, retroviruses, herpes viruses and lentiviruses.

The gag, pol and env genes transfected packaging cell line being introduced with retrovirus as vector. This leads to virus particle having the RNA of the foreign gene, capable of infecting a cell only once. This retroviral vector infects the host cell (once), leads to insertion of the non host DNA into host genome. This may/may not express the non host (foreign) gene depending upon its location on the genome. Gene therapy can be attempted in either germ cells or somatic cells.

(d) 3. Non-viral gene delivery:

There are three main non-viral vector systems:

1. Cationic liposomes;

2. DNA-polymer conjugates; and

3. Naked DNA.

Non-viral vectors have the inherent property to bypass some of the specific problems noticed with viruses’ i.e. immune response, limited packaging capacity, and random integration. Gene delivery systems based on cationic liposome have been tested in cystic fibrosis.

(d). 4. Microinjection:

A solution of DNA of interest is inserted into each cell directly using ultra fine bore needles. There is one limitation with this i.e. it can be done in very little number of cells. However, large number of cells can be targeted using viruses or chemical treatments.

(d). 5. Chemical and physical methods:

Gene transfer can be accomplished by exposing huge number of target cells with an amalgam of DNA and chemicals. DNA is infused into the cells via fusion with the outer membrane of target cells thereby, letting the contents introduced into the cells.

The cell fusion is comparatively simpler, and larger numbers of cells can be considered for treatment. The main concern is the uncertainty i. e. DNA infusion in some of the cells further some will let it get inserted it into their genome later very few may actually facilitate its expression.

Electrical treatment is also employed to induce cellular uptake of DNA (electroporation). Albeit vector is not required in chemical and physical methods, it has two major limiting factors:

1. The rate of incorporation of DNA in to the cellular genome is usually very much limited i.e. 1:10000 to 1:100000, However, large number of cell needs to be treated and the cells that take up and incorporate the required DNA should essentially be separated from other cells.

2. The DNA inserts at random and in multiple copies into the cell’s genome and it is therefore relatively uncontrolled and unpredictable.

The liposomes can be engineered from specifically treated cells like RBC or bacteria. It is, similar to chemical treatment therefore remains nonspecific at delivery and pretty unpredictable in outcome.

(d) 6. Targeted gene correction:

This is a radical new technology for correcting genetic defects also known as chimeraplasty. It is a non-viral method of gene therapy developed by Eric Kmiec (1996). Chimera last is the synthetically prepared molecule including of both RNA and DNA.

Chimeraplasty has the potential to repair single base pair mutations, deletions, or insertions in DNA. The chimeraplast is designed with nucleotide sequences complementary to the base pair of gene having defect. The chimeraplast enters in to a cell, introduce itself into the nucleus. Further it attaches to the correct gene adjacent to malfunctioning gene and fail to bind properly at’ defective sites. The cell’s repair system acts like zip, reads this bump in the DNA, consider it as a signal to repair the defective gene.

(d) 7. Germ line gene therapy:

This technique involves the genetic modification of germ cells. The genetic constitution of egg/sperm could be attempted to change by such therapy. Although sperms are tiny and tough to penetrate besides being in vast number therefore may not be feasible to modify at gene level. The egg cells are greater in size therefore can relatively be handled easily.

The ova/egg cells may be considered for correction just before fertilization. Early embryonic cell has been subjected to germ line therapy. However, insertion of new genetic information into gametes is theoretically possible. Genetic alteration of sperm producing cell may be a better option. This may not thoroughly facilitate genetic changes in sperm precursors and all sperm ultimately.

Germ line correction would lead to genetic alteration that could be detected in all cells in the body and could be passed on to next generations if the recipient is allowed to reproduce. It has drawn severe ethical concern, therefore considered unacceptable as well due to safety reasons.

(d) 8. Somatic cell gene therapy:

In somatic cell gene therapy, treatment affects only cells in the patients’ organs, excluding germ line cells. This does not lead to inheritance as it would not be passed on to the offspring. The genetic information is encrypted in to the specific sequence of nucleotides in its DNA. DNA ultimately is the thread which acts as band master of all of the substances that comprise and regulate the cell. Specific sequences of DNA contain quanta of information for specific proteins such as enzymes, hemoglobin, or the number of receptors.

The piece of DNA that contains the information intended for a specific product is called genes. The DNA of the gene will be same for somatic or germ line therapy, but there is chance of difference in sequences of nucleotides added/conformed adjacent to the gene depending on the pattern of gene regulation in a particular treatment.

The intention of somatic gene therapy is to rectify these genetic defects. Recessive genetic traits usually act as vehicle via which genetic diseases are passed on to next generation. Such diseases are usually fatal in early part of life, if not treated. The very motto of this therapy is to facilitate insertion of piece of DNA in to somatic cell DNA. This may

facilitate restoration of function of enzyme to produce normal protein. Early embryonic cells or Germ cells may be attempted to correct a genetic defect. This would affect/ rectify somatic cells and germ line cells in the body.

Clinical experiments in gene therapy should supposedly be performed on somatic cells of patients to attempt limited correction of life-threatening diseases, This might alleviate the signs and symptoms due to single gene defect. The consideration of gene therapy would be depending upon nonexistence of any alternative-treatment. This would be depending upon several factors, also on animal experimentations.

Gene transfer may not be reliable enough. This may be benefiting straight way to the subject. There are limitations of somatic cell therapy especially in the disorders that affect multiple tissues, as cells of each organ would have to be altered.

It would not be effective for those tissues composed of cells that are not dividing, such as brain and muscle. Correcting germ line cells are unpredictable. If a defect is rectified, it would be more likely to have corrected descendants. But there is chance of getting things in adverse line as well.

Essay on Alternative Treatments:

Hemochromatosis can be allayed by periodic blood donation. Dietary measures for PKU, galactosemia and urea cycle defects improves prognosis among human, though only partially effective. Wernicke-Korsakoff encephalopathy responds to Vitamin supplementation. Drug treatments can compensate for some genetic defects.

There are drugs that can be partially helpful in sickle cell disease by inducing expression of a type of hemoglobin, usually during fetal development. Hemophilia may be countered by administration of clotting factors. Innovation in the areas of drug delivery i.e. pumps that reside in the body and deliver hormones, enzymes, or other chemicals for long periods of time may reduce the need for gene therapy. Some genetic defects may be rectified by transplantation of whole organs or tissues. Bone marrow transplantation has been used in treating thalassemia, sickle cell disease, and immune deficiencies.

There is thus several existing and prospective treatment for genetic diseases that do not require direct gene replacement or supplementation, but all have limitations and many genetic diseases have no treatment. However, the therapy of most genetic disorders is still ineffective and inadequate. Gene therapy of somatic cells will therefore probably prove technically superior to alternative treatments for selected patients with some disorders.

Gene therapy has got limited success in stead of its earlier roller coaster ride of excitement and hope to failure and disappointment. It has been assumed to be helpful in different diseases such as severe combined immunodeficiency, cancer, growth hormone deficiency, cystic fibrosis, cardiovascular and neurological diseases, duchenne muscular dystrophy, influenza, malaria, H3V infection, diabetes mellitus, sickle cell disease, hemophilia, factor IX deficiency, Lesch-Nyhan disease and ADA and PNP deficiencies, PKU, galactosemia, Wernicke-Korsakoff encephalopathy etc.

Prognosis:

The patient expected quality and length of life directly affect the potential benefit and acceptable level of risk of any medical or experimental intervention. Extremely serious disorders, such as Lesch-Nyhan disease and ADA and PNP deficiencies, have such poor prognoses that even small potential benefits are welcome and large risks may be acceptable to the patient and his or her family in comparison to continued life with the disease.

Future Prospects:

Opinions vary as some believes that gene therapy presents realistic opportunities for new ways to treat cancer, but not for the next 10-15 years. Others do not expect that working remedies for a wide-range of diseases will emerge in future.

However, it is opined that research will contribute to the knowledge of vectors, insertion and delivery techniques, and safety. Such understanding will be critical to the development of potential gene therapies in future. Eventually, it may be predicted that in the distant future gene therapy could be used to produce customized animals with more muscle and milch ability and whatever traits we may desire.

One big challenge for gene therapies is delivering DNA or RNA safely to cells inside patients' bodies. New nanoparticles could be an improvement over the current standard – repurposed viruses.

A successful gene therapy for the leading cause of sight loss in children is being reviewed by the FDA.

The gene therapy CTL019 induced complete remission in 90% of patients.

A new research paper reports dangerous side effects in CRISPR-edited mice. Some scientists are pushing back, placing blame for the unwanted mutations on the experiment, not the technique.

Researchers are starting to harness the potential of this much-hyped gene editing technique – with coming applications in medicine, biology and agriculture.

A report released by the US National Academies of Science and Medicine underscores the potential of gene editing and acknowledges the sensitivities in managing the ethical dimensions.

Cystic fibrosis (CF) affects around 3,000 people in Australia and 70,000 worldwide. It's an inherited disease caused by a mutation in a single gene called CFTR.

How new therapies could prove a headache for the world of sport.

A toddler from London has had gene therapy for leukaemia and it seems to have worked. But why have breakthroughs taken so long?

Until more is understood, it's sensible to limit experimentation that would make changes to germ line cells that would be passed on to future generations.

Gene therapy is allowing us to switch on natural beneficial mutations to counteract the effects of negative mutations in diseases such as sickle cell anaemia.

That genetic editing techniques have become as straightforward as they have poses questions for how we want them to be used.

When you buy a diet coke, or any other consumables containing the artificial sweetener aspartame, you’ll see a warning against consuming the product if you have phenylketonuria, an inherited metabolic…

Viruses have traditionally been mankind’s enemies, causing disease and often mutating out of the reach of our medicines. But now a new technology is conscripting them into doing good. Viral vectors show…

Increasing the activity of enzyme NEU1 can stop or reverse the build-up of plaque in the brains of mice with Alzheimer’s…

Every now and again you might read about gene therapy and efforts to correct serious genetic diseases. But I’m betting that very few readers have had gene therapy, nor have they ever met anyone who has…

The presence of foreign objects, like viruses, in our bloodstream is usually a bad thing. Evolution has created some extremely efficient immune cells that patrol the blood, seeking out material that should…

Genetic traits like a bulbous nose or balding give some people reasons to moan about what they inherited from their parents. But more serious genetic flaws can cause debilitating disease. Now, Italian…

The new treatment of inherited blindness in dogs might help restore vision to humans too. A combined study lead by Michigan…

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