Gene Therapy of Human Disease

Table of Contents 1. Introduction 2. Methods of Transfer 2.1. Naked DNA transfer 2.2. Viral gene transfer 2.2.1. Adenoviruses 2.2.2. Retroviruses 2.2.3. Lentiviruses 2.2.4. Adeno-associated viruses 2.2.5. Herpes simplex viruses 2.2.6. Other viruses 2.3. Nonviral gene transfer 2.3.1. Chemical and physical methods 2.3.2. Liposomal gene transfer 2.3.3. Protein/peptide gene transfer 3. Disease Targets for Gene Therapy 3.1. Genetically determined protein deficiencies 3.1.1. Gaucher disease 3.1.2. Adenosine deaminase deficiency 3.1.3. γc Cytokine receptor deficiency 3.1.4. Cystic fibrosis 3.2. Gene therapy for acquired diseases 3.2.1. Immune stimulation in the treatment of cancer 3.2.2. Activation of ganciclovir by herpes thymidine kinase 3.2.3. Tumor suppressor genes 3.2.4. Acquired immunodeficiency syndrome (AIDS) 3.2.5. Rheumatoid arthritis 3.2.6. Cardiovascular disease 4. Conclusions 1. Introduction The treatment of genetic diseases has seemed to many a daunting challenge. What, after all, can be done if the immutable basic blueprints of the body have a serious imperfection? In reality, even in the middle of the last century it was possible to improve greatly the quality of life and, indeed, to save the lives of patients with some such genetic diseases. The successful approaches included dietary manipulation as in phenylketonuria or galactosemia, surgery to correct various deformities, and sometimes avoidance of inciting factors in the environment, as in acute intermittent porphyria. However, until the latter part of the century just past, the possibility of actually changing the faulty genetic blueprints was beyond the imagination of the realistic medical scientist. In 1972 Friedmann and Roblin (68) argued persuasively that defective genes could be replaced by those with the correct sequence. In the following 30 years a great deal of effort has been expended to bring such a therapeutic endeavor to fruition, thus far with limited success. 2. Methods of Transfer Although it has been possible to achieve expression of genes delivered to the cytoplasm (28,37,100,119) the most common and useful strategy is to deliver the gene of interest to the nucleus. There are 4 extracellular barriers to such delivery: 1) opsonins, 2) phagocytes, 3) extracellular matrices, and 4) degradative enzymes (59). An opsonin is a factor that attaches to foreign material and renders it more susceptible to ingestion by phagocytes. Thus, opsonins can inactivate a gene delivery system by attachment, leading to the inactivation of the gene and/or its carrier. Phagocytes are cells that can inactivate a gene delivery system by engulfment or digestion. The extracellular matrix represents a physical barrier of polymerized protein and carbohydrates present between cells protecting target cells from relatively large DNA carrier systems. Finally, the extracellular fluid contains DNases that can rapidly digest unprotected DNA. Once all these barriers are overcome, the gene delivery system attaches to the plasma membrane where it faces cellular barriers. The first of these is the plasma membrane. Then the gene must be protected from nucleases in the cytoplasm and overcome the possibility of endosomal entrapment. The nucleic acid must enter the nucleus where the gene of interest can proceed to be transcribed and translated, and finally the protein traffics to the cellular location where it has a function. The ideal gene delivery vector is nontoxic, nonimmunogenic, easy to produce in large quantities, and efficient in protecting and delivering DNA into cells, preferably with a specificity for a particular cell type. This ideal vector remains to be discovered (112,185). Properties of existing transfection agents are compared in Table 1.TABLE 1: Properties of transfection agents*2.1. Naked DNA transfer The principal obstacle to cellular DNA uptake is charge (56). In an aqueous solution, such as the milieu that bathes cells in the body, DNA has a net negative charge. DNA tends to be repelled from cell membranes, because they, too, are negatively charged. There are a few exceptions where cells appear to be able to assimilate naked DNA; this includes the successful target protein expression after direct muscular injections in mice (22,38,56,181). While the mechanism of this type of gene transfer is unclear, a small amount of tissue damage or increased pressure at the injection site may play a role (56). A few of cells and can be by the direct injection of naked DNA these the cells and cells Other of cells are to transfection a carrier is Naked in is and must be into a delivery or vector of efficient into cells Naked DNA DNA by are in and, by and the the Naked DNA can be delivered the of with a gene or the The gene a pressure to deliver DNA into the the pressure for delivery into 2.2. Viral gene transfer have for cellular and and these has been a in to achieve efficient transfer of genes to Viral gene delivery has been a of and herpes simplex 2.2.1. Adenoviruses are for gene transfer to cell are DNA viruses is of and the and viruses are relatively easy to produce in large and a large for foreign to all genes and the for the of most genes a are by the inactivation of the of protein or DNA or by the inactivation of the gene that has and with of the and genes can of to in is for in cell gene delivery to the of the target gene of interest the and it a that this type of gene delivery for The to from and to of cells This is with to and cellular with a cells The of of and a The of a in an gene therapy has a of the and of The was a with an of in 4 after a been into the few have or by all genes and with by viruses In some was such to mice at with viruses have been In with of have been for gene as in the of viruses and for is to produce of and to by There is some that of are this possibility 2.2.2. Retroviruses a of viruses is transcribed into DNA in the The basic contains genes as and these genes are by as the and the of the and are for the of the as Viral can the of a or can be in with the The contains a that the to be from in the cell Retroviruses the of genes into for in to this of gene transfer is the that into the leading to the or inactivation of genes to the of the In rapidly this has to the of that and Retroviruses can with cellular or DNA or to produce viruses or the of that after the of cells in of a gene transfer the of viruses the gene of the with the of the vector to a that in The vector in this has been replaced by a of of with the of as as have been with of that is are as vector is inactivation by the In DNA to more expression with these that cells have the to and inactivate DNA are protected by that the expression of or The of DNA into a can of the that DNA the of gene have been to to the of the to achieve of delivering genes to target cells with specificity of the can be the of the the and the The target is a receptor or a can be for that have site that with the and a that with the cell the can a of by that or for receptor more susceptible to is to by a the a factor target cells until with factor in possible with the of of and the of to from vector The of cells is by the of or of from such as that This is to be to of the cell 2.2.3. are immunodeficiency that have the of into the of and cells can be to cell to most cells, such as However, as the most are the of these must be can be these must be to be to or with to produce an The cells to produce be in the contains the and In it genes for and have the to cell damage or in can produce an acquired immunodeficiency syndrome disease if in mice The gene the of to This gene has been replaced with from viruses that have a for from to many cells, viruses have a the is and the this contains that can the of and cellular The injection of has been to in expression for in and cells, the inactivation of expression as is in 2.2.4. Adeno-associated Adeno-associated viruses are DNA that genes into of and cells are to disease in the of have to have and that are between the and the of the and the The the of the The gene the or the gene is in and In the of the gene into a of a in this site specificity to be the of has been replaced with the gene to be viruses the genes that to gene therapy are limited because can transfer small foreign genes and vector is to the of the gene and some of the viruses are to produce this can be with that have been for of in a with or muscular injections of an vector factor the factor in A of in has been The as a of fibrosis because have and to those of the The of this that successful gene transfer in this is with DNA transfer in of patients for at 2.2.5. Herpes simplex Herpes simplex type is vector that has been for its to transfer genes to is a relatively large that contains more genes of the genes of in the life genes that can be to in cell the cells of the of where it can a as an in a to as from of in and of In the of even in the of defective for and genes are and are are for in and many and vector are rapidly after injection into the has been in the of defective with The of as a vector for gene transfer is limited because many have been by this and to of those from all are have relatively of a of the gene produce 2.2.6. Other The and viruses are viruses that are as possible The from these viruses deliver to the cytoplasm of target cells where can produce large of protein from of the gene of interest into the and thus produce foreign protein or DNA are for of of DNA at the The system is for in gene therapy because of its as a its large and the of its The contains a a and and a the expression of genes the cytoplasm of cells is a DNA as a vector for gene has a the that many and it can and cells has been in and it or cells was an of of the with of some is in and it into cellular its must be it can be as a vector for gene transfer 2.3. Nonviral gene transfer Nonviral gene transfer with an of efficient gene delivery to in of The of gene therapy is to the successful for cellular barriers that efficient expression of the target gene the with gene The of a vector could to the cell endosomal to the and, in some into the target cell The of gene delivery is the transfer of from the to the nucleus (56). In this are by cells into the endosomal The of this with its be to rapidly (56). is to of and has been in some gene therapy to endosomal of DNA 2.3.1. Chemical and physical have been to DNA with such as and the to DNA and it to be more by Chemical are for gene methods such as and are relatively for gene the of an that the cell DNA to enter the cytoplasm for and the have been to overcome some of the barriers to efficient gene transfer of the of In has an that is to be a in its efficient gene transfer In transfection by has been In this are with that to cell can in the cells the are by transfection of cells with DNA or The are a and the cells are to tissue for This has the of cell and it cells to a of The that this of cells as determined by and that it in cells 2.3.2. Liposomal gene are of a membrane an aqueous The net charge of the type and of the with DNA at in part an with the negatively charge of DNA The in can DNA (59). Liposomal gene transfer has of of and the to large DNA While this of gene transfer has useful the of to DNA must be to the of the mechanism of DNA delivery and DNA remains to be In have a limited of delivery and gene and have with negatively target cells such as to the of can be as and into the to the cellular and of at the cell or the endosomal 2.3.3. Protein/peptide gene with DNA in protein and gene that is between the and and the negatively in the DNA The of and have been to DNA and to in cellular uptake of of DNA and are DNA with of and are (59). DNA that are in gene transfer and the protein and with to be to and in gene transfer with or as as those with or more DNA a and produce large as determined by at that with of DNA and that in from to as the increased The of an as a by a to the of in a in and of transfection as determined by in cells as compared with There is to and DNA and with these as as the of the of such (59). Once DNA has been the of gene delivery can be by the of into the may target cells a cell gene delivery a and a have been a of and an of gene transfer the cellular of for Other to gene delivery with the following 1) to 2) to endosomal and 3) agents to with factors that the after into the milieu (59). have to with gene therapy have that for a and a endosomal have a of gene transfer by An of DNA transfection DNA and was in in a The transfection of and in with compared with have been to the role of acid in the of membrane protein is a acid with the following with and at a is a acid to for the of acid and with the acid in A A A the is a to an into more and it can of and membrane is a that to membranes, and DNA transfection The acid of is a protein that of the of the factor to the cell of the gene The of gene transfer in has been limited by the of these in to DNA and the of agents to DNA by a of gene delivery agents has been into was that the of more the of that can be in the cell in gene expression was with the its DNA may as the is to a that could and DNA The in of these has to be The of gene transfer of and the to target nucleic to cell the for of and the of the or type of the nucleic acid that can be The for efficient gene delivery is the of the the between and charge are to the for In to the methods of gene and gene transfer the of a delivery can be and and that could to the for the and expression of nucleic In DNA the and its in many Once are can be to a with to those of the last few a of have in gene However, in of the interest in this is a of the mechanism of of and the of this of gene In in of have been such as are to be and to have the of and in gene transfer the of an 3. Disease Targets for Gene Therapy Gene therapy can correct genetic disease the of a The of gene therapy is to or the of to can overcome some with the of protein of and the last more patients have been in gene transfer of target genetic diseases Gaucher disease deaminase deficiency immunodeficiency disease and fibrosis Gene therapy has therapeutic in the treatment of acquired diseases such as cancer arthritis and disease The of gene therapy are in Table of gene therapy Genetically determined protein deficiencies 3.1.1. Gaucher Gaucher disease is a disease by the deficiency of the is as an and the most common of disease and the target cell in this disease is the transfer of the gene has been in in of patients with Gaucher disease cells have been with a the and to in the mice a The from this that can cells in the with that the and that can be to after transfer of the gene into cells from patients with Gaucher disease in the in of cells 3.1.2. Adenosine deaminase Adenosine deaminase the of and to and in the The deficiency of this is the of immunodeficiency an of the system by and The of the system to in patients with deficiency is to the of or to the of to the into cells and in patients years the gene to be in and cells, and in of the and of cellular and cells from the as a target cell in a of of transfection to be for a of In a in and cell in an to produce of transfection The to these of can sometimes a The with a to of that can be with The was into the that the site was at the location of the site in the In to the of expression from in target cell for or included from and in that the of the and the cell the of in the of the and the cell was for cell these are to the for has as a for the of the and of gene therapy with cells have been to and and the can in with a possible in for the of is and the gene is in Thus, is a to of the gene in cells, and to the of 3.1.3. γc Cytokine receptor is an by γc receptor deficiency that to an in and γc gene expression as as and cell can be with gene transfer in transfection of cells in patients with the γc in γc and cells, as as and cell to those of of the patients and of with a was to and the of and and In this γc gene transfer was or This the of diseases for gene therapy in the cell has a The of this the first that in gene therapy can be to genetic disease 3.1.4. Cystic Cystic fibrosis is a disease from in the gene the fibrosis this protein a that is by and by The of fibrosis is defective by by and In the of gene transfer as a treatment for was it was that gene transfer of the the in cells The relatively for that of the of for direct to where the of cells by of The of is limited because cell and is to have a In the direct transfer of the gene to in was a vector the and the of the and the was for to transfection A gene transfer was in 4 patients with A vector the was to the and of these patients of this that a and syndrome of was this was to be an to the after of A of gene transfer in patients patients with disease A vector the by a was delivered to the of the patients in this in 4 that of and This was a in the patients the the of gene transfer by a the of 4 of patients gene transfer by a at the of patients of cells by the with of or of the limited the of that could be delivered to overcome the of gene In a of as a of gene transfer of to was in of to the A of the of was and to be with a to by In of the transfer of the to the of patients was in 4 patients of patients of gene and patients to of the Finally, of the transfer of the to the of patients was where patients DNA was in of the and of in 3.2. Gene therapy for acquired diseases 3.2.1. Immune stimulation in the treatment of are to the of gene transfer as cancer most of these the to cancer expression or such as is that of in be of leading to and a stimulation may the with The an by the of cells after or of the first of a cancer to of In this a that in the of was into cells as a with with the cells protected a with the cell The that the of a in a a the of and cells This has been to in the is the transfer of in patients with The vector in this was of the and it and in genes able to cells with an vector to and cells with at and for a of 4 injections if the first the disease of patients was of patients to the patients to therapy with a of by the in of cells into has in a of for in with has in some patients with and cell of and is a of to that cells to 3.2.2. Activation of ganciclovir by herpes thymidine gene transfer strategy is the in genetic of cells with the delivery of herpes simplex thymidine kinase the of ganciclovir following thymidine cells The ganciclovir is by to a and to the of a ganciclovir The of into DNA to and and cell cells In the of from the ganciclovir system is be from the of gene it is to the of the cellular to cells, and has been the of the gene therapy is a in patients with the of is to the and of A vector the gene the of the was into the of these by of therapy with from to was of patients gene 3.2.3. Tumor suppressor Tumor suppressor genes for that to cells that have genetic damage by or the of such genes may in is a of that a role in cellular and In its role as of the it cell at the by the of genes that cell into it in to DNA and it DNA genetic in the of the suppressor gene are to be in to of A the of gene transfer in patients with cancer therapy patients to injections of the from to In of the vector DNA was by patients for disease in this in disease was in the patients to have 3.2.4. Acquired immunodeficiency syndrome The of gene therapy of is the of the cellular with cells to possible target cell is the that could a of and cells, such as cells, and these cells are useful as are in is a protein that is for as a between the nucleus and cytoplasm to the of and this to the of the to the of in and cells from cells a to the and of in transfer of a gene into cells from genes are the of the The cells into the with of While the transfection was to be between and the after all The of this the for gene transfer An have an vector for gene transfer by as many genes and as of for and to This vector with to factors for efficient transfection and This vector can cell the cells are by The of is in cells by this by for for for the treatment of disease that to the of a target this type of vector could to of A to a by and is a of the to deliver a of that it in membranes, to be in the of This of and it cells its cell and the DNA. The DNA is transcribed by the to produce that an for to and cells with the 3.2.5. Rheumatoid Rheumatoid arthritis is a disease of that is by by with of cells In and The cellular that this the and factor and cell for The has been with and receptor in and of The was a of and as as the of the of and that may play a role in arthritis have been for gene therapy of the first is therapy that genes and the is therapy in the gene are to the Gene therapy could as a delivery system of agents to the of these are or by is a to therapy is and has that may its with A by the system with the of that the system is of cells in the The protein to the of and the of was that of into arthritis of these cells in and in in as a therapy for arthritis gene delivery of an receptor has been in a of arthritis is an that the of to its cell the of arthritis in delivery of protected from arthritis and was in the treatment of as as in the of arthritis in 3.2.6. Cardiovascular has been gene transfer the that a role in its and have thus to cellular Cardiovascular gene transfer can of the delivery of genes to in the by A is to deliver genes that cell for by with DNA cell or cell after or a The expression of factor is by and various expression and cell and it In and factor as a for cells such as cells and and have been in and in to The of gene transfer to after has been in a and A of patients in this a with a with the and an as a In all the was in the of between treatment and In a the of to the of in of patients with to disease in patients with and/or Naked DNA the acid of was into the of the and DNA was with and Gene expression was with by a in after gene in of in to or improve in 4 of and successful possible in patients for the The of this treatment was limited to in patients with of has been to be a useful for patients with A was in patients with and therapy injections of DNA the acid of in the The injections into a The of all patients after gene was in all patients The of this treatment was limited to at the of with as as The direct injection of into is a and useful in patients with 4. Conclusions is that in gene therapy the of many to the disease that is approaches that in expression of the in some Gaucher expression may to material that has many In some such as cell a gene be and it be to the of the In some acquired such as expression of a gene may be to the In such as expression may be The of and the of tissue expression from to Thus, the of gene therapy of the of the gene that is of the and of the disease that is more is the to from the and of a treatment with an Gene therapy is the of and a of cell and is to all the and of of these The first in the of have been by the that have the the and by for gene therapy of interest to gene therapy as a strategy to correct in DNA. The of this strategy is that the most of is between an DNA vector and the as the of the gene remains in and genetic the of this and to the possibility of in the of are in in The is to that of the target gene with the that it contains a in the DNA with the DNA sequence. The is as it is to be by the DNA of the gene the that are This strategy has been successful in the in of the factor gene in has been to correct a in the in a

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