Gene therapy

1
Gene therapy

• Any procedure to treat or alleviate disease which
  involves genetically modifying the cells of the
  patient
• can be used to treat
• infectious diseases
• cancers
• inherited disorders
• immune system disorders
• Scientific hurdles in gene therapy
• concept of vehicles called vectors (gene
  carriers) to deliver therapeutic genes to the
  patients' cells
• Once the gene is in the cell, it needs to operate
  correctly
• Patients' bodies may reject treatments
• need to regulate gene expression.

2
Gene therapy approaches

• Classical
• Deliver genes too appropriate target cells with
  aim of obtaining optimal expression
• Once inside target cells, genes may
• Produce a product the patient lacks (loss of
  function mutations)
• Kill diseased cells directly (cancer cells)
• Activate immune system to kill cells
• Non-classical
• Inhibit expression of genes associated with
  pathologies (gain-of-function mutations, for
  example)
• Correct a genetic defect and restore normal gene
  expression (targeted gene replacement or
  correction)

3
Somatic versus germline therapy

• Somatic
• Genetic defect is only corrected in somatic ells
  of affected individual
• Any genetic changes are restricted to treated
  person
• Few ethical concerns
• Germline
• Genetic modifications made to the gamete,
  fertilized egg or embryo
• Any stable changes will be passed on to all
  future generations
• Used to create transgenic mice
• Research into such techniques is currently
  prohibited in most countries
• Not the same as cloning!

4
in vivo versus ex vivo therapy
5
Different tissues are targeted

• Ex vivo
• Suitable when affected tissues can be removed,
  altered genetically and reintroduced
• Works best for blood cells or skin cells
• Used to treat ADA deficiency
• In vivo
• Cloned genes are transferred directly into
  tissues
• When cells cannot be cultured in vitro (brain
  cells, lung epithelia)

6
Gene addition versus replacement

• Ideally gene therapy would aim to replace
  defective genes with normal copies
• Involves homologous recombination and selection
  to detect small percentage of cells that have
  made change
• Would not be useful for in vivo therapy
• Germline therapy would require precise gene
  replacement- major technological hurdle
• New techniques are being developed for gene
  replacement using recombination or gene
  correction
• Gene addition most common technique in somatic
  therapy
• Transgene works along side of mutated gene
• Cannot correct a dominant mutation

7
gene addition

• Gene addition seeks to compensate for a defective
  gene (red) by providing cells with a corrective
  gene (green). Genes can be injected directly into
  cells, or they can be coaxed in by chemical or
  electrical means. Most delivery systems deposit
  corrective genes in the cells nucleus, where it
  remains only transiently. Other methods integrate
  genes into the chromosomes. Integrated genes can
  be passed on to progeny cells in the course of
  normal cell division, which may provide long-term
  therapeutic benefits.

8
Vehicles/vectors for gene transfer

• Various techniques using viral vectors have been
  developed
• They each have advantages and disadvantages based
  on the list below
• Factors to consider
• Efficiency of delivery to target cell
• Specificity of delivery
• Expression of introduced gene in target cell
• Dividing versus non-dividing
• Size of DNA that can be carried
• Stability of gene in target cell
• Induction of host immune response?

9
Retroviral vectors

• RNA viruses that encode reverse transcriptase
• Infect cells, viral RNA genome ? DNA, integrates
  into genome into a single site on host chromosome
• Very efficient at transferring DNA and integrated
  DNA is very stable
• Richard Mulligan and Constance Cepko, at MIT,
  made the important technological leap that
  initiated the modern era of gene therapy
• members of the retrovirus family could be
  engineered to carry foreign genes into mammalian
  cells and splice them into the hosts
  chromosomes.

10
Retroviruses for gene therapy

• Murine leukemia virus (MLV)
• Integration requires LTRs
• Pol reverse transcriptase ? required for
  packaging of virus Env viral envelope protein
  Gag viral core protein
• virus needs to be unable to replicate

11
Control of gene expression in MLV

• When constructing their retroviral vectors,
  Mulligan and colleagues opted to use promoters
  native to the virus, rather than the corrective
  genes own promoter. In laboratory petri dishes,
  these vectors sometimes worked quite well, but
  not always.
• In some cases, the therapeutic genes entered the
  cells as expected but were expressed at
  unpredictably low levels. Low levels of
  expression continue to dog gene-therapy efforts,
  and improving expression levels remains a major
  focus of research. Recent vectors include
  portions of the genes own promoter. This has the
  added benefit that the therapeutic gene is
  expressed as naturally as possible -- only during
  the times when its product is needed.

12
Controlling retroviral vectors

• Certain promoters are sensitive to tetracycline
  and are activated when the drug is present. A
  vector was recently constructed by Herman Bujold
  and colleagues at the University of Heidelberg
  that pairs a tetracycline-sensitive promoter with
  a corrective test gene. The test gene would be
  activated only if the patient ingests
  tetracycline.
• More recently, retroviral vectors have been
  developed that can be infused directly into an
  organ, such as the liver, or placed into the lung
  by inhalation.

13
Drawbacks to retroviral gene therapy

• Retroviruses only infect continually
  proliferating cells
• certain blood cells (not stem cells)
• Cells lining gastrointestinal tract
• Other cell types divide but discontinuously, or
  never divide (mature neurons)
• Can be a useful way to target cancer cells
• Size of DNA is limited (7 kb)
• Does not specifically target one cell type
• New techniques aimed at Env protein
• Fuse a protein hormone to Env so virus is
  targeted to cells expressing that hormone
  receptor
• Integrates at random sites in the genome- may
  cause insertional mutagenesis
• If integration occurs near a proto-oncogene, may
  cause activation

14
Adenoviral vectors

• Double-stranded DNA viruses that infect the
  non-dividing cells of the respiratory and
  gastrointestinal tracts
• Once inside cells, replicate as episomes (do not
  integrate)
• To disable the virus, the E1 gene is replaced
  with foreign DNA(6 kb)
• The bulk of the early work on adenoviral gene
  therapy was conducted by Ronald Crystal at
  Cornell Medical School and James Wilson at the
  University of Pennsylvania.
• Major drawbacks
• The adenoviral vectors have to be repeatedly
  administered in order to maintain a steady
  therapeutic dose.
• some of the vectors cause a strong inflammatory
  response at the high levels required to achieve
  therapeutic doses
• have been used in human trials to correct
  mutations in the CFTR gene. The success of these
  trials, however, has been quite low. For one
  thing, the hosts immune system registers the
  adenoviral vector as foreign and eliminates it
  from the system.

15
Adeno-associated virus (AAV)

• Adeno-associated virus (AAV) are non-pathogenic
  human parvoviruses
• Dependant on a helper virus, usually adenovirus,
  to proliferate
• Infects a wide range of cells, including lung and
  muscle cells, and integrates its genes at a
  specific site within the hosts chromosome 19.
• Can infect non-dividing cells and does not elicit
  an immune response -- both of which are important
  advantages over retroviral and adenoviral
  vectors.
• Integration into the host genome allows prolonged
  transgene expression.
• Gene transfer into vascular epithelial cells,
  striated muscle hepatic cells has been
  reported, with prolonged expression when the
  transgene is not derived from a different
  species.
• There has been no attempt to target particular
  cell types other than by localised vector
  delivery.
• Pioneered by three investigators Kenneth Berns
  and Nicholas Muzyczka at the University of
  Florida and R. Jude Samulski at the University of
  North Carolina at Chapel Hill.

16
AAV

• The wild type genome is a single stranded DNA
  molecule, consisting of two genes rep, coding
  for proteins which control viral replication,
  structural gene expression integration into the
  host genome, cap, which codes for capsid
  structural proteins. At either end of the genome
  is a 145 bp terminal repeat (TR), containing a
  promoter
• a cDNA and promoter can be inserted to replace
  rep and cap, but must be smaller than 4.7 kb

17
AAV and protein delivery

• Used two AAVs
• Loaded one with the gene for erythropoietin, or
  Epo, a recombinant protein that stimulates red
  blood cell production and is used to treat
  anemias
• Loaded the other with the genes for a
  transcription factor complex able to regulate
  Epo.
• T particular transcription factor can be switched
  on by a small-molecule drug called rapamycin,
  which can be taken orally.
• Significantly, the amount of rapamycin
  administered controlled the level of Epo produced
  by cells exposed to the AAV vectors.
• giving more rapamycin stimulated a proportionate
  rise in production of Epo, resulting in higher
  numbers of red blood cells in the bloodstream.
  Conversely, stopping administration of rapamycin
  shut down production of Epo. The effects of the
  treatment were tracked for six months in mice and
  three months in monkeys.
• Epo was chosen as a demonstration gene for the
  new drug delivery system because it is a
  therapeutically significant drug that currently
  must be given by injection several times a week
  and because its action can easily be measured
  through red blood cell counts.

18
Herpesvirus

• Tropic for the central nervous system
• Can establish lifelong latent infections in
  neurons
• Have large insert size capacity (20 kb)
• Non-integrating
• Long-term expression of gene is impossible
• Goal is to use these for treatment of
  neurological diseases such as Parkinson's and for
  treating CNS tumours
• Currently not being actively used

19
Comparison of vectors
20
Non-viral DNA transfer

• Non-viral methods of DNA transfer
• require only a small number of proteins
• have a virtually infinite capacity
• have no infectious or mutagenic capability
• large scale production is possible using
  pharmaceutical techniques.
• Methods of non-viral DNA transfer
• Liposomes and lipoplexes
• naked DNA
• Receptor-mediated endocytosis

21
Liposomes and lipoplexes

• Spherical vesicles composed of synthetic lipid
  bilayers which mimic the structure of biological
  membranes

22
Liposomes

• In vitro up to 90 of certain cell lines may be
  transfected.
• By including a small amount of an anionic lipid
  in an otherwise cationic liposome the DNA can be
  incorporated into the internal surface of the
  liposome, thus protecting it from enzymatic
  degradation.
• The inclusion of a DNA-binding protein enhances
  transcription by bringing the plasmid into the
  nucleus.
• Further proposed improvements include
  incorporating Epstein-Barr genes in the plasmid
  to maintain the plasmid as an episomal element.
• No limit to DNA size
• Efficiency of transfer is low and DNA does not
  integrate
• Expression of inserted genes is transient

23
Naked DNA

• Was discovered by accident when naked DNA was
  used as a negative control in liposome expts
• Naked DNA (in the form of a plasmid) can be
  directly injected into muscle cells or attached
  to gold particles that are bombarded into the
  tissue (gene guns)
• Injection has been used to target muscle cells in
  mice with mdx defect, model for Duchenne
• DNA believed to enter the cell through small
  lesions in cell membrane
• Not very efficient, but can result in prolonged
  low level expression in vivo if stably
  integrated.
• could be useful in cells that dont deivide
  frequently, like muscle cells

24
Receptor-mediated endocytosis

• DNA coupled to a targeting molecule that can bind
  to a specific cell surface receptor
• Covalent linkage of polylysine to receptor (
  charge)
• Bind DNA (- charge)
• Transferrin receptor expressed in many cell
  types, enriched in proliferating cells and blood
  cells
• After endosome formation, some DNA can migrate to
  nucleus, but most is degraded- not integrated
• Can also be used with cell-specific receptors
  like asialoglycoprotein receptors, found on
  surface of hepatocytes and are used for clearing
  ASGP from serum
• Complexes would be infused into liver and taken
  up by specific cells

25
Targeted inhibition of gene expression

• Selectively inhibit the expression of a specific
  gene in vivo
• Well suited to cancer and infectious disease
• May offer hope of treating dominantly inherited
  diseases, to specifically inhibit expression of
  mutant allele
• Most obvious desire
• Targeted recombination for gene replacement
• Still very ineffcicient
• Various other approaches
• Targeted inhibition of expression at DNA level
• Targeted inhibition of expression at RNA level
• Targeted inhibition of expression at protein
  level
• One more approach- in vivo DNA repair

26
Targeted inhibition of expression at DNA level

• DNA can form a triple helix if bound by a
  specific oligonucleotide
• Such binding will prevent transcription of the
  targeted gene

27
Targeted inhibition of expression at RNA level

• Anti-sense therapeutics to block expression of
  disease-causing genes
• Binding of gene-specific oligodeoxynucleotides
  (instead of oligoribo-) or anti-sense transcripts
  to RNA
• May involve a catalytically active RNA (ribozyme)
  that binds to RNA and cleaves it, rendering it
  inactive
• Antisense oligodeoxynucleotides (ODN) can be
  synthesized simply and transferred into cells
  with liposomes
• Will not bind to double stranded DNA, specific
  for RNA
• ODN-RNA hybrids sensitive to attack from RNAseH,
  causes actual destruction of mRNA

28
Targeted inhibition of expression at protein level

• Intracellular antibodies (intrabodies)
• Can be directed to a specific cell compartment
  where they can bind to and inactivate
  disease-causing molecules
• Oligonucleotide aptamers
• Oligonucleotides that can bind to specific
  protein sequences
• Identify by screening thousands of oligos for
  binding
• Idea is to transfer large amounts of oligos into
  specific cell to block protein function
• No therapeutic function found yet
• Mutant proteins
• Proteins often form multimeric complexes
• Design poison proteins to inactivate proteins
  needed for the life cycle of a pathogen, like AIDS

29
In vivo correction of a mutation

• To treat certain disorders that are not easily
  treated by other means
• Uses synthetic chimeric DNA/RNA molecules to
  induce site-directed repair in vivo
• way to repair damaged genes, rather than
  replacing them.
• Takes advantage of cells DNA repair mechanism
• place into the cell a small hybrid RNA-DNA
  molecule called a chimeric oligomer that pairs
  with the defective gene in the region of the
  error.
• Repair enzymes use the oligomer as a template to
  guide the correction. The oligomer binds snugly
  with the defective gene except in the region of
  the error, where the mismatch causes a bulge.
• Repair enzymes detect this bulge and replace the
  erroneous nucleotides. In this example the
  guanine (G)-cytosine (C) pair is incorrect. The
  oligomer provides the template indicating that an
  adenine (A)-thy mine (T) pair should be inserted
  in that spot. The repair enzymes follow the
  instructions in the template and correct the gene
  accordingly. Corrections made this way endure for
  generations of cell divisions.

30
Gene correction

• successfully corrected seven chromosomal targets
  with this approach.
• feasibility of using gene repair to correct the
  sickle-cell mutation in vitro
• correction is maintained through successive
  generations of cell division, suggesting that
  gene repair may have long-term benefits.

31
Gene therapy trials underway
32
Gene therapy to treat cancer

• gene therapy has emerged as a potential
  alternative to the existing treatments, and its
  potential is such that protocols for the
  treatment of cancer now account for over 80 of
  the gene therapy clinical trials .
• General approaches
• Stimulate natural killing of cancer cells
  (enhance immunogenicity)
• Artificial killing of cancer cells (toxin)
• Inhibit expression of oncogenes (DNA, RNA,
  protein levels)
• Gene addition of tumour suppressors

33
Immunotherapy

• The aim of immunopotentiation is to enhance the
  response of the immune system to cancers, thereby
  leading to their destruction.
• Passive immunotherapy aims to increase the
  pre-existing immune response to the cancer whilst
  active immunotherapy initiates an immune response
  against an unrecognized or poorly antigenic
  tumour.
• Passive immunotherapy usually involves harvesting
  tumour infiltrating lymphocytes treating them
  to express increased cytokines e.g. IL-2
  TNF-alpha. The cell population is then expanded
  in vitro returned to the patient.
• Tumour cells are used for active immunotherapy,
  genetically modifying them to increase expression
  of antigen presenting molecules/co-stimulatory
  molecules (e.g. B7), local concentrations of
  cytokines (e.g. IL-2) or tumour antigens (erbB2
  oncoprotein).

34
Immunopotentiation

• The cells are then irradiated prior to being
  returned to the patient, preventing the
  reintroduction of replication competent tumour
  cells. These approaches have been termed cancer
  vaccines.
• Increasing the immunogenicity of tumours may then
  lead to an anti-tumour response. Because immunity
  is a systemic reaction, this immune reaction
  could potentially eliminate all the tumour cells
  in the body, including sites of metastatic
  deposit.

35
Immune approaches
36
Gene-Directed Enzyme Pro-drug Therapy

• Introduction of genes that encode enzymes capable
  of converting pro-drugs to cytotoxic drugs is the
  basis of the GDEPT approach to cancer gene
  therapy.
• A relatively harmless pro-drug can be
  administered to a patient following the
  transfection of some tumour cells with genes
  encoding enzymes that will activate the pro-drug
  in situ to form a cytotoxic drug that will kill
  the tumour cell.
• This approach may be considered as using gene
  therapy to improve upon conventional
  chemotherapy. The local expression of an
  activating enzyme ensures that the peripheral
  toxicity often associated with chemotherapy is
  reduced. The use of a relatively harmless
  pro-drug ensures that high doses can be
  administered to the patient, resulting in high
  concentrations of the cytotoxic drug being
  produced in vicinity of the tumour.
• Following the death of the tumour cell, the
  cytotoxic drug may be able diffuse into
  neighbouring cells and kill them, a phenomenon
  known as the bystander effect. The bystander
  effect ensures that it is not necessary to
  transfect all of the cells in the tumour, indeed
  it has been shown that 100 cell death can be
  achieved in vitro following transfection of only
  10 of the cells.

37
Suicide Gene Therapy or molecular chemotherapy

• E. coli cytosine deaminase (CD) gene 5-
  fluorocytosine (5-FC).
• CD converts 5-FC to 5- FU, a chemotherapeutic
  agent.
• This combination produces a bystander effect and
  has been demonstrated to have some success in
  animals with hepatic metastasis of
  gastro-intestinal tumors.
• Delivery of CD to specific sites and the use of
  tissue specific promotors are a focus of work
  with this strategy.
• Herpes simplex virus thymidine kinase gene
  (HSV-tk) ganciclovir (GCV).
• HSV/TK converts the prodrug ganciclovir into
  toxic metabolites, that inhibit the synthesis of
  DNA.
• Also causes a bystander effect. This strategy has
  been looked at for treatment of localized brain
  tumors, liver metastases.
• Unpredictability of bystander effect and
  difficulties in transduction have kept cure rates
  low.
• The use of tissue-specific vectors may improve
  the efficacy of the approach in time.

38
Tumour suppressor gene therapy

• Goals of tumor suppressor gene therapy
• cell death
• changes in
• growth of the cell
• behavior of the cell
• invasiveness of the cell
• metastatic ability of the cell.
• Because p53 is the most common gene mutated in
  cancer and influences transcription, cell cycle
  movement, apoptosis, and angiogenesis, it is a
  prime target for gene replacement.
• In model systems, transduction of cancer cells
  with p53 has been demonstrated to
• inhibit growth,
• inhibit angiogenesis, and
• induce apoptosis.
• Early clinical trials using a p53 retrovirus have
  also been encouraging.

39
Prostate cancer therapy and p53

• Hypothesis effective transfer of the p53 gene
  would result in cancer cell death, ultimately
  resulting in reduction of tumor size.
• A total of 26 men with advanced localized
  prostate cancer who were candidates for a
  prostatectomy participated in the Houston study.
  Each man received an injection containing a
  synthetic form of the p53 gene, or an
  "adenoviral-p53 gene". The injection went
  directly into the prostate tumor.
• The size of the tumor was monitored with
  ultrasound over a six-week period. Subsequently,
  all of the men received a prostatectomy, after
  which the size of the prostate tumor was
  measured. Results of the study showed that seven
  of the men, or 27 percent of the study
  participants, experienced at least a 25-percent
  reduction in the size of the tumor. In addition
  to reduction in tumor size, the investigators saw
  increased cell death and intracellular transfer
  of the p53 protein, evidence of the potential
  efficacy of the gene therapy.

40
Gene therapy for inherited disorders
41
Beginnings of gene therapy trials for heritable
diseases

• The first human trials of gene therapy began in
  1990 using a strategy of ex vivo gene therapy.
• The first therapeutic trials utilizing the ex
  vivo approach attempted to treat two genetic
  disorders, including children with an inherited
  form of immune deficiency adenosine deaminase
  deficiency with severe combined immuno-deficiency
  (ADA-SCID), as well as children and adults with
  extremely high levels of serum cholesterol.
• The first model for in vivo gene therapy was
  based on an attenuated version of the adenovirus
  in the treatment of cystic fibrosis. Adenoviruses
  have a natural tropism for lungs in that they are
  associated with respiratory diseases.

42
ADA trial

• 5-6 mo. after beginning the therapy the T cell
  counts in patient 1 rapidly increased until they
  reached the normal range where they have
  remained. The levels of the ADA enzyme were also
  found to have increased significantly.
• Patient 2 also showed an increase in the number
  of T cells, but no significant increase in ADA
  levels could be detected.

43
2002- success in treating SCID

• 18 month old boy in England
• bone marrow cells removed and mixed with a mouse
  retrovirus carrying normal version of defective
  gene
• Cells later transfused back into patient
• Now he has a normal immune system
• A second child that started therapy later is
  doing even better

44
Cystic fibrosis gene therapy

• primary defect is in lungs- no way too culture-
  must use in vivo therapy- aerosol to deliver
  vector
• First trial (1993) used adenovirus complications
  with dosage
• More recent trials- liposome-mediated transfer of
  the gene encoding CFTR into nasal airway
  epithelial cells.
• Expression of the plasmid DNA was detectable in
  all 9 patients who received liposome treatment
  (through detection of RNA) and there was partial
  restoration of the electrophysiological defect.
• In most cases the response to low chloride
  perfusion was restored to about 20 of that seen
  in non-CF subjects, although in one patient the
  response to low chloride perfusion was within the
  normal range for non-CF subjects.
• The maximal effect occurred 3 days after exposure
  to the liposome/DNA complexes, but reverted to
  pre-treatment levels 7 days after exposure.
• As well as demonstrating detectable gene
  expression in patients, this study also showed
  that there were no adverse clinical effects and
  nasal biopsies showed no adverse histological or
  immunological changes.
• CF gene therapy remains ineffective at present

45
Gene therapy- hypercholesterolemia

• Familial hypercholesterolemia (FH)- dominant
  deficiency of LDL receptor
• People with this inherited condition have
  dangerously high blood levels of cholesterol, in
  spite of their body weight or diet.
• results from a defective gene that encodes a
  receptor found on the membranes of liver cells
  specific for low-density lipoprotein (LDL)bad
  cholesterol
• Normally LDL enters liver cells via this
  receptor, after which the liver clears the body
  of LDL. But people with FH have too few
  functioning receptor molecules and cannot remove
  LDL from their blood. As a result, blood serum
  levels of LDL are too high in people with this
  condition, and many FH patients develop coronary
  artery disease.
• In animal models, investigators demonstrated some
  success when corrective copies of the receptor
  gene were transferred into liver cells via a
  retroviral vector.
• Human therapy- found hepatocytes can be cultured
  in vitro, and infused back into patient where
  they re-seed liver
• Very invasive procedure

46
Familial hypercholesterolemia

• The experience of one 28-year-old woman
  represents one of the better outcomes of this
  clinical trial.
• The patient lacked any detectable functioning LDL
  receptor (homozygous mutant)
• At the start of the trial, she had 482 milligrams
  of LDL in each deciliter (mg/dl) of blood, well
  over twice the normal level of 160 to 210 mg/dl.
• Her liver cells were then treated with a
  retroviral vector containing the LDL-receptor
  gene. Within a few days, her serum cholesterol
  dropped by 180 mg/dl to about 300 mg/dl.
• With additional cholesterol-lowering drugs, her
  LDL blood levels stabilized at around 356 mg/dl
  and remained there for about two and a half
  years. These levels, although lower than they
  were originally, are still higher than they ought
  to be.

47
Therapy for DMD

• many difficulties in delivery
• Initial positive results with mouse model using
  modified myoblasts have not been repeated with
  humans
• Gene therapy approaches
• Retroviral vectors cant be used because adult
  muscle fibers are non-dividing
• Adenoviral vectors have been used for in vivo
  delivery to muscle fibers, but do not provide
  necessary continued expression
• Size of dystrophin gene is limiting- cDNA 14 kb
• Alternative method
• Induce expression of dystrophin-related gene
  (utrophin) using gene therapy or drugs that
  stimulate utrophin expression

48
Limb girdle muscular dystrophy (1999)

• Gene therapy approaches to treat muscular
  dystrophy have been hampered by an inability to
  successfully place the therapeutic genetic
  material into deficient muscle cells.
• using a naturally-occurring hamster model of
  LGMD, researchers have developed a technique that
  successfully produces widespread transfer of
  corrective genetic material into muscle cells
  throughout an entire limb
• In LGMD, the instability of muscle tissue is
  linked directly to the level of genetic
  disruption that occurs within the sarcoglycan
  complex - a critical muscle structure composed of
  four membrane-spanning proteins. Depending on the
  nature of the mutations that can affect any one
  of these proteins, the integrity of the muscle
  membrane is compromised - eventually resulting in
  muscle weakness that can range from a very mild
  form to a more severe, rapidly-progressing type
• problem how best to gain access to the millions
  and millions of muscle cells that require genetic
  re-engineering. Dismissing intramuscular
  injection as an impractical technique (since
  literally thousands of injections might be
  needed), they proposed an intravascular route
  that would require some means of allowing infused
  genetic material (being carried in
  adeno-associated viruses) to seep out of the
  blood vessels into the surrounding muscle tissue.

49
Limb girdle muscular dystrophy (1999)

• The viruses are millions of times bigger than
  oxygen molecules and too big to leave the blood
  vessels under normal circumstances.
• The solution- make the blood vessels become
  leaky, temporarily, so that the viruses could
  leave the vessels and make contact with nearby
  muscle tissue.
• Thus, histamine - a natural vessel destabilizer
  -- was added to the liquid solution carrying the
  adeno-associated viruses.
• Infusing just a single limb - a leg - with the
  histamine-enhanced solution, scientists were able
  to achieve widespread gene transfer to all the
  muscles in that particular area of the body.
• A 36-year-old man became the first person to
  receive a gene therapy injection for muscular
  dystrophy.
• Injected a muscle on the top of his foot with
  genes for a muscle protein.
• One of Decker's foot muscles received the
  therapeutic genes, while the same muscle of the
  other foot received a sham injection. Researchers
  will take biopsies to compare the condition of
  the two muscles after six weeks.

50
OTC deficiency

• studies carried out at Institute for Human Gene
  Therapy (IHGT), formed in 1993 and headed by Dr.
  Jim Wilson
• A deficiency of the urea cycle enzyme Ornithine
  Transcarbamylase (OTC) is a paradigm for
  metabolic diseases.
• Genes deficient in this enzyme are unable to
  break down nitrogen, which leads to an
  accumulation of such toxic substances as ammonia.
  Investigators were recently approved to evaluate
  the utility of gene therapy for treating OTC
  deficiency.
• When infused into the blood this recombinant
  virus targets the liver cells very specifically
  and efficiently.
• Patients eligible for this clinical trial are
  adults partially deficient of OTC. This would
  include the affected males who live beyond
  adolescence and adult carrier females.
• Participating patients undergo a procedure in
  which a catheter is inserted into a groin artery
  and advanced into the vessel that perfuses the
  liver.

51
OTC trial setback

• Eighteen-year-old Jesse Gelsinger, a participant
  in the experimental gene therapy trial for
  ornithine transcarbamylase (OTC) deficiency, died
  four days after being injected with corrective
  genetic material. 
• Jesse was the 18th patient to participate in the
  Phase I clinical trial, which began in April of
  1997 as a means to develop an effective treatment
  for OTC deficiency - an inherited disorder that,
  in its most common form, causes death in affected
  newborn males because of their inability to
  properly process nitrogen in food proteins due to
  a genetic defect in the liver. 
• None of the 17 other trial participants who
  preceded Jesse in the OTC trial developed any
  serious unexpected clinical responses to the gene
  therapy protocol
• The findings suggest that the experimental drug
  used in the trial initiated an unusual and deadly
  immune-system response that led to multiple organ
  failure and death.