Recombinant DNA and Gene Cloning

Recombinant DNA is DNA that has been created artificially. DNA

from two or more sources is incorporated into a single recombinant
molecule.

Making Recombinant DNA (rDNA): An Overview

 Treat DNA from both sources with the same restriction

endonuclease (BamHI in this case).
 BamHI cuts the same site on both molecules

5' GGATCC 3'

3' CCTAGG 5'

 The ends of the cut have an overhanging piece of single-

stranded DNA.
 These are called "sticky ends" because they are able to base pair
with any DNA molecule containing the complementary sticky
end.
 In this case, both DNA preparations have complementary sticky
ends and thus can pair with each other when mixed.
 a DNA ligase covalently links the two into a molecule of
recombinant DNA.
To be useful, the recombinant molecule must be replicated many
times to provide material for analysis, sequencing, etc. Producing
many identical copies of the same recombinant molecule is called
cloning. Cloning can be done in vitro, by a process called the
polymerase chain reaction (PCR). Here, however, we shall examine
how cloning is done in vivo.

Cloning in vivo can be done in

 unicellular microbes like E. coli

 unicellular eukaryotes like yeast and
 in mammalian cells grown in tissue culture.

In every case, the recombinant DNA must be taken up by the cell in a

form in which it can be replicated and expressed. This is achieved by
incorporating the DNA in a vector. A number of viruses (both
bacterial and of mammalian cells) can serve as vectors. But here let us
examine an example of cloning using E. coli as the host and a
plasmid as the vector.
Plasmids

Plasmids are molecules of DNA

that are found in bacteria separate
from the bacterial chromosome.

The desirable properties are:

 are small (a few thousand

base pairs)
 usually carry only one or a
few genes
 are circular Electron micrograph of an E. coli
 have a single origin of cell ruptured to release its DNA.
replication The tangle is a portion of a single
Plasmids are replicated by the DNA molecule containing over
4.6 million base pairs encoding
same machinery that replicates the
approximately 4,300 genes. The
bacterial chromosome. Some
plasmids are copied at about the small circlets are plasmids.
(Courtesy of Huntington Potter
same rate as the chromosome, so a
and David Dressler, Harvard
single cell is apt to have only a
´plasmids are copied at a high rate Medical School.)
and a single cell may have 50 or more of them.

Genes on plasmids with high numbers of copies are usually expressed

at high levels. In nature, these genes often encode proteins (e.g.,
enzymes) that protect the bacterium from one or more antibiotics.

Plasmids enter the bacterial cell with relative ease. This occurs in
nature and may account for the rapid spread of antibiotic resistance in
hospitals and elsewhere. Plasmids can be deliberately introduced into
bacteria in the laboratory transforming the cell with the incoming
genes.
PLASMID CLASSIFICATION.

The most useful classification of naturally occurring plasmids is based

on the main characteristic coded by the plasmid genes. The 5 main
types of plasmids according to this classification are:

1. Fertility plasmids″ F″: Fertility plasmids carry only tra

genes(transfer) and have no characteristic beyond the ability to
promote conjugal transfer of plasmids.

2. Resistant″ R″ plasmids: They carry genes conferring on the host

bacterium resistance to one or more antibacterial agent such as
chloramphenicol, ampicillin and mercury. R plasmids are very
important in clinical microbiology as they are spread through
natural populations and can have profound consequence in the
treatment of bacteria infections. Example RP4.

3. Col plasmids:They code for colicins. These colicins are proteins

that kill other bacteria e.g. colE1 of E.Coli.

4. Degradative plasmids: They allow the host bacterium to

metabolise unusual molecules such as Toluene and Salicylic
acid e.g. TOL of Plasmodium putida.

5. Virulence plasmids: These confer pathogenicity on the host

bacteriume.g. Ti plasmids of agrobacterium tumefaciens, which
induce Crown Gall disease on dicotyledonous plants.

An Example:

pAMP

 4539 base pairs

 a single replication origin
 a gene (ampr)conferring resistance to the antibiotic ampicillin
(a relative of penicillin)
 a single occurrence of the sequence
 5' GGATCC 3'
3' CCTAGG 5'

that, as we saw above, is cut by the restriction enzyme BamHI

 a single occurrence of the sequence

5' AAGCTT 3'

3' TTCGAA 5'

that is cut by the restriction enzyme HindIII

Treatment of pAMP with a mixture of BamHI and HindIII produces:

 a fragment of 3755 base pairs carrying both the ampr gene and
the replication origin
 a fragment of 784 base pairs
 both fragments have sticky ends

pKAN

 4207 base pairs

 a single replication origin
 a gene (kanr) conferring resistance to the antibiotic kanamycin.
 a single site cut by BamHI
 a single site cut by HindIII

Treatment of pKAN with a mixture of

BamHI and HindIII produces:

 a fragment of 2332 base pairs

 a fragment of 1875 base pairs with the
kanr gene (but no origin of replication)
 both fragments have sticky ends

These fragments can be visualized by

subjecting the digestion mixtures to
electrophoresis in an agarose gel. Because of its negatively-charged
phosphate groups, DNA migrates toward the positive electrode
(anode) when a direct current is applied. The smaller the fragment, the
farther it migrates in the gel.

Ligation Possibilities
If you remove the two restriction enzymes and provide the conditions
for DNA ligase to do its work, the pieces of these plasmids can rejoin
(thanks to the complementarity of their sticky ends).

Mixing the pKAN and pAMP fragments provides several (at least 10)
possibilities of rejoined molecules. Some of these will not produce
functional plasmids (molecules with two or with no replication origin
cannot function).

One interesting possibility is the joining of

 the 3755-bp pAMP fragment (with ampr and a replication

origin) with the
 1875-bp pKAN fragment (with kanr)
Sealed with DNA ligase, these molecules are functioning plasmids
that are capable of conferring resistance to both ampicillin and
kanamycin. They are molecules of recombinant DNA.

Because the replication origin, which enables the molecule to function

as a plasmid, was contributed by pAMP, pAMP is called the vector.

Transforming E. coli

Treatment of E. coli with the mixture of

religated molecules will produce some
colonies that are able to grow in the
presence of both ampicillin and
kanamycin.

 A suspension of E. coli is treated with the mixture of religated

DNA molecules.
 The suspension is spread on the surface of agar containing both
ampicillin and kanamycin.
 The next day, a few cells — resistant to both antibiotics — will
have grown into visible colonies containing billions of
transformed cells.
 Each colony represents a clone of transformed cells.

However, E. coli can be simultaneously transformed by more than

one plasmid, so we must demonstrate that the transformed cells have
acquired the recombinant plasmid.

Electrophoresis of the DNA from doubly-resistant colonies (clones)

tells the story.
  Plasmid DNA from cells that acquired their resistance from a
recombinant plasmid only show only the 3755-bp and 1875-bp
bands (Clone 1, lane
3).
 Clone 2 (Lane 4) was
simultaneous
transformed by
religated pAMP and
pKAN. (We cannot
tell if it took up the
recombinant molecule
as well.)
 Clone 3 (Lane 5) was transformed by the recombinant molecule
as well as by an intact pKAN.

Cloning other Genes

The recombinant vector described above could itself be a useful tool

for cloning other genes. Let us assume that within its kanamycin
resistance gene (kanr) there is a single occurrence of the sequence
5' GAATTC 3'
3' CTTAAG 5'
This is cut by the restriction enzyme EcoRI, producing sticky ends.

If we treat any other sample of DNA, e.g., from human cells, with
EcoRI, fragments with the same sticky ends will be formed. Mixed
with EcoRI-treated plasmid and DNA ligase, a small number of the
human molecules will become incorporated into the plasmid which
can then be used to transform E. coli.

But how to detect those clones of E. coli that have been transformed
by a plasmid carrying a piece of human DNA?

The key is that the EcoRI site is within the kanr gene, so when a
piece of human DNA is inserted there, the gene's function is
destroyed.
All E. coli cells
transformed by
the vector,
whether it carries
human DNA or
not, can grow in
the presence of
ampicillin. But E.
coli cells
transformed by a
plasmid carrying
human DNA will
be unable to grow
in the presence of
kanamycin.

So,

 Spread a
suspension of treated E. coli on agar containing ampicillin only
 grow overnight
 with a sterile toothpick transfer a small amount of each colony
to an identified spot on agar containing kanamycin
 (do the same with another ampicillin plate)
 Incubate overnight

All those clones that continue to grow on ampicillin but fail to grow
on kanamycin (here, clones 2, 5, and 8) have been transformed with a
piece of human DNA.

Some recombinant DNA products being used in human therapy

Using procedures like this, many human genes have been cloned in E.
coli or in yeast. This has made it possible — for the first time — to
produce unlimited amounts of human proteins in vitro. Cultured cells
(E. coli, yeast, mammalian cells) transformed with a human gene are
being used to manufacture more than 100 products for human therapy.
Some examples:
  insulin for diabetics
 factor VIII for males suffering from hemophilia A
 factor IX for hemophilia B
 human growth hormone (HGH)
 erythropoietin (EPO) for treating anemia
 several types of interferons
 several interleukins
 granulocyte-macrophage colony-stimulating factor (GM-
CSF) for stimulating the bone marrow after a bone marrow
transplant
 granulocyte colony-stimulating factor (G-CSF) for
stimulating neutrophil production (e.g., after chemotherapy) and
for mobilizing hematopoietic stem cells from the bone marrow
into the blood.
 tissue plasminogen activator (TPA) for dissolving blood clots
 adenosine deaminase (ADA) for treating some forms of severe
combined immunodeficiency (SCID)
 parathyroid hormone
 many monoclonal antibodies
 hepatitis B surface antigen (HBsAg) to vaccinate against the
hepatitis B virus
 C1 inhibitor (C1INH) used to treat hereditary angioedema.

The Polymerase Chain Reaction (PCR):

Cloning DNA in the Test Tube
The polymerase chain reaction is a technique for quickly "cloning" a
particular piece of DNA in the test tube (rather than in living cells like
E. coli). Thanks to this procedure, one can make virtually unlimited
copies of a single DNA molecule even though it is initially present in
a mixture containing many different DNA molecules.
The procedure

 In order to perform PCR, you must know at least a portion of the

sequence of the DNA molecule that you wish to replicate.
 You must then synthesize primers: short oligonucleotides
(containing about two dozen nucleotides) that are precisely
complementary to the sequence at the 3' end of each strand of
the DNA you wish to amplify.
 The DNA sample is heated to separate its strands and mixed
with the primers.
 If the primers find their complementary sequences in the DNA,
they bind to them.
 Synthesis begins (as always 5' -> 3') using the original strand as
the template.
 The reaction mixture must contain
o all four deoxynucleotide triphosphates (dATP, dCTP,
dGTP, dTTP)
o a DNA polymerase. It helps to use a DNA polymerase that
is not denatured by the high temperature needed to
separate the DNA strands.
 Polymerization continues until each newly-synthesized strand
has proceeded far enough to contain the site recognized by the
other primer.
 Now you have two DNA molecules identical to the original
molecule.
 You take these two molecules, heat them to separate their
strands, and repeat the process.
 Each cycle doubles the number of DNA molecules.

Using automated equipment, each cycle of replication can be

completed in less than 5 minutes. After 30 cycles, what began as a
single molecule of DNA has been amplified into more than a billion
copies (230 = 1.02 x 109).
With PCR, it is routinely possible to amplify enough DNA from a
single hair follicle for DNA typing. Some workers have successfully
amplified DNA from a single sperm cell. The PCR technique has
even made it possible to analyze DNA from microscope slides of
tissue preserved years before. However, the great sensitivity of PCR
makes contamination by extraneous DNA a constant problem.
 Genetic Recombination in Bacteria
Bacteria have no sexual reproduction in the sense that eukaryotes do.
The have

 no alternation of diploid and haploid generations

 no gametes
 no meiosis

But the essence of sex is genetic recombination, and bacteria do have

three mechanisms to accomplish that:

 transformation
 conjugation
 transduction

Transformation

Many bacteria can acquire new genes by taking up DNA molecules

(e.g., a plasmid) from their surroundings. The ability to deliberately
transform the bacterium E. coli has made possible the cloning of
many genes — including human genes — and the development of the
biotechnology industry.

The first demonstration of bacterial transformation was done with

Streptococcus pneumoniae and led to the discovery that DNA is the
substance of the genes. The path leading to this epoch-making
discovery began in 1928 with the work of an English bacteriologist,
Fred Griffith.

The cells of S. pneumoniae (also known as the pneumococcus) are

usually surrounded by a gummy capsule made of a polysaccharide.
When grown on the surface of a solid culture medium, the capsule
causes the colonies to have a glistening, smooth appearance. These
cells are called "S" cells.
Streptococcus pneumoniae
(pneumococci) growing as
colonies on the surface of a
culture medium. Left: The
presence of a capsule around
the bacterial cells gives the
colonies a glistening, smooth
(S) appearance. Right:
Pneumococci lacking capsules
have produced these rough (R)
colonies. (Courtesy of Robert
Austrian, J. Exp. Med. 98:21,
1953.)

However, after prolonged cultivation on artificial medium, some cells

lose the ability to form the capsule, and the surface of their colonies is
wrinkled and rough ("R"). With the loss of their capsule, the bacteria
also lose their virulence. Injection of a single S pneumococcus into a
mouse will kill the mouse in 24 hours or so. But an injection of over
100 million (100 x 106) R cells is entirely harmless.

Encapsulated
(left) and
nonencapsula
ted (right)
pneumococci
. The
encapsulated
forms
produce
smooth
colonies
(above).
(Courtesy of
Robert
Austrian, J.
Exp. Med.
 98:21, 1953.)

The reason? The capsule prevents the pneumococci from being

engulfed and destroyed by scavenging cells — neutrophils and
macrophages — in the body. The R forms are completely at the mercy
of phagocytes.

Pneumococci also occur in over 90 different types: I, II, III and so

on. The types differ in the chemistry of their polysaccharide capsule.

Unlike the occasional shift

of S -> R, the type of the
organism is constant. Mice
injected with a few S cells
of, say, Type II

pneumococci, will soon

have their bodies teeming
with descendant cells of the
same type.

However, Griffith found that when living R cells (which should have
been harmless) and dead S cells (which also should have been
harmless) were injected together, the mouse became ill and living S
cells could be recovered from its body. Furthermore, the type of the
cells recovered from the mouse's body was determined by the type of
the dead S cells. In the experiment shown, injection of

 living R-I cells and

 dead S-II cells

produced a dying mouse with its body filled with living S-II
pneumococci.

The S-II cells remained true to their new type. Something in the dead
S-II cells had made a permanent change in the phenotype of the R-I
cells. The process was named transformation.
Oswald Avery and his colleagues at The Rockefeller Institute in New
York City eventually showed that the "something" was DNA.

In pursuing Griffith's discovery, they found that they could bring

about the same kind of transformation in vitro using an extract of the
bacterial cells.

Treating this extract with

 enzymes to destroy all polysaccharides (including the

polysaccharide of the capsule)
 a lipase to destroy any lipids
 proteases to destroy all proteins
 RNase to destroy RNA

did not destroy the ability of their extracts to transform the bacteria.

But treating the extracts with DNase to destroy the DNA in them did
abolish their transforming activity. So DNA was the only material in
the dead cells capable of transforming cells from one type to another.
DNA was the substance of genes.

View an electron micrograph showing DNA entering a

pneumococcus.

Although the chemical composition of the capsule is determined by

genes, the relationship is indirect. DNA is transcribed into RNA and
RNA is translated into proteins. The phenotype of the pneumococci
— the chemical composition of the polysaccharide capsule — is
determined by the particular enzymes (proteins) used in
polysaccharide synthesis.

Conjugation

Some bacteria, E. coli is an example, can transfer a portion of their

chromosome to a recipient with which they are in direct contact. As
the donor replicates its chromosome, the copy is injected into the
recipient. At any time that the donor and recipient become separated,
the transfer of genes stops. Those genes that successfully made the
trip replace their equivalents in the recipient's chromosome.
Features:

 Can only occur between cells of opposite mating types.

+
o The donor (or "male") carries a fertility factor (F ).
−
o The recipient ("female") does not (F ).
 F
o is a set of genes originally acquired from a plasmid and
now integrated into the bacterial chromosome;
o establishes the origin of replication for the chromosome.
o A portion of F is the "locomotive" that pulls the
chromosome into the recipient cell.
o The rest of it is the "caboose".
 In E. coli, about one gene gets across each second that the cells
remain together. (So, it takes about 100 min for the entire
genome (4377 genes) to make it. But,
 the process is easily interrupted so
o it is more likely that host genes close behind the leading F
genes ("locomotive") will make it than those farther back
o The "caboose" seldom makes it so failing to receive a
complete F factor, the recipient cell continues to be
"female"
 The DNA that makes it across finds the homologous region on
the female chromosome and replaces it (by a double crossover).
 By deliberately separating the cells (in a kitchen blender) at
different times, the order and relative spacing of the genes can
be determined. In this way, a genetic map — equivalent to the
genetic maps of eukaryotes — can be made. But here the map
intervals are seconds, not centimorgans (cM).

Demonstration

The figure shows the mechanism of

conjugation in E. coli cells where

 The "male" lacks functional

genes needed to synthesize the
vitamin biotin and the amino
acid methionine (Bio−, Met−)
 so these must be added to its culture medium.
 The "female" has those genes (Bio+, Met+) but has nonfunctional
(mutant) genes that prevent it from being able to synthesize the
amino acids threonine and leucine (Thr−, Leu−) so these must be
added to its culture medium).
 When cultured together, some female cells receive the
functional Thr and Leu genes from the male donor.
 A double crossover enables them to replace the nonfunctional
alleles.
 Now the cells now can grow on a "minimal" medium containing
only glucose and salts.

Transduction

Bacteriophages are viruses that infect bacteria. In the process of

assembling new virus particles, some host DNA may be incorporated
in them.
The virion head can hold only so much DNA so these viruses

 while still able to infect new host cells

 may be unable to lyze them.

Instead the hitchhiker bacterial gene (or genes) may be inserted into
the DNA of the new host, replacing those already there and giving the
host an altered phenotype. This phenomenon is called transduction.

Significance of genetic recombination in bacteria.

Transformation, conjugation, and transduction were discovered in the

laboratory. How important are these mechanisms of genetic
recombination in nature? We don't really know, but

Some thoughts:

 The completion of the sequence of the entire genome of a

variety of different bacteria (and archaea) suggest that genes
have in the past moved from one species to another. This
phenomenon is called lateral gene transfer (LGT).
  The remarkable spread of resistance to multiple antibiotics may
have been aided by the transfer of resistance genes within
populations and even between species.
 Many bacteria have enzymes that enable them to destroy foreign
DNA that gets into their cells. It seem unlikely that these would
be needed if that did not occur in nature. In any case, these
restriction enzymes have provided the tools upon which the
advances of molecular biology and the biotechnology industry
depend.

Reductionism

The understanding of complex systems almost always has to await

unraveling the details of some simpler system. You may feel that
trying to find out how one type of pneumococcus could be converted
into another was an exceedingly specialized and esoteric pursuit. But
Avery and his coworkers realized the broader significance of what
they were observing and, in due course, the rest of the scientific world
did as well. By electing to work with a well-defined system: the
conversion of R forms of one type into S forms of a different type,
these researchers made a discovery that has revolutionized biology
and medicine.

Attempting to understand the workings of complex systems by first

understanding the workings of their parts is called reductionism.
Some scientists (and many nonscientists) question the value of
reductionism. They favor a holistic approach emphasizing the
workings of the complete system.

But the record speaks for itself. From skyscrapers to moon walks, to
computer chips to the advances of modern medicine, progress comes
from first understanding the properties of the parts that make up the
whole.

The late George Wald, who won the 1967 Nobel Prize in Physiology
or Medicine for his discoveries of the molecular basis of detecting
light [Link], once worried that his work was overly specialized —
studying not vision, not the eye, not the whole retina, not even their
rods and cones, but just the chemical reactions of their rhodopsins.
But he came to realize "it is as though this were a very narrow
window through which at a distance one can see only a crack of light.
As one comes closer, the view grows wider and wider, until finally
through this same window one is looking at the universe. I think this
is the way it always goes in science, because science is all one. It
hardly matters where one enters, provided one can come closer...."

Gene Therapy I
Many human diseases are caused by defective genes.

A few common examples:

Disease Genetic defect
hemophilia A absence of clotting factor VIII
cystic fibrosis defective chloride channel protein
muscular dystrophy defective muscle protein (dystrophin)
sickle-cell disease defective beta globin
hemophilia B absence of clotting factor IX
any one of several genes fail to make a
severe combined
protein essential for T and B cell
immunodeficiency (SCID)
function

All of these diseases are caused by a defect at a single gene locus.

(The inheritance is recessive so both the maternal and paternal copies
of the gene must be defective.) Is there any hope of introducing
functioning genes into these patients to correct their disorder?
Probably.

Other diseases also have a genetic basis, but it appears that several
genes must act in concert to produce the disease phenotype. The
prospects of gene therapy in these cases seems far more remote.

Case study: severe combined immunodeficiency (SCID)

SCID is a disease in which the patient has neither

 cell-mediated immune responses nor

  is able to make antibodies.

It is a disease of young children because, until recently, the absence of

an immune system left them prey to infections that ultimately killed
them.

About 25% of the cases of SCID are the result of the child being
homozygous for a defective gene encoding the enzyme adenosine
deaminase (ADA). The normal catabolism of purines is deficient, and
this is particularly toxic for T cells and B cells.

Treatment Options:
 Raise the child in a strictly germfree environment: all food,
water, and air to be sterilized. David, the "bubble boy" from
Houston, survived this way until he was 12 years old.
 Give the child a transplant of bone marrow from a normal,
histocompatible, donor. Ideally, this would give the child a
continuous source of ADA+ T and B cells. However,
o even though the child cannot reject the transplant (the
child has no immune system), T cells in the transplant
(unless the donor was an identical twin) can attack the
cells of the child producing graft-versus-host disease.
o the donor cells may be infected with a virus which could
overwhelm the recipient before his or her immune system
was restored. (David received a bone marrow transplant
from his sister, but she, like many people, had been
infected earlier with the Epstein-Barr virus (the cause of
"mono")). The virus was still present in the cells she
donated, and killed her brother.
 Give injections of ADA (the enzyme is currently extracted from
cows). When conjugated with polyethylene glycol (PEG) to
delay its breakdown in the blood, ADA-PEG injections have
kept SCID patients reasonably healthy. But just like the insulin
injections of a diabetic, they must be repeated at frequent
intervals. So,
 what about giving the patient functioning ADA genes; that is,
gene therapy?
Gene Therapy: requirements

 The gene must be identified and cloned.This has been done for
the ADA gene.
 It must be inserted in cells that can take up long-term residence
in the patient. So far, this means removing the patient's own
cells, treating them in tissue culture, and then returning them to
the patient.
 It must be inserted in the DNA so that it will be expressed
adequately; that is, transcribed and translated with sufficient
efficiency that worthwhile amounts of the enzyme are produced.

All these requirements seem to have been met for SCID therapy using
a retrovirus as the gene vector. Retroviruses have several advantages
for introducing genes into human cells.

 Their envelope protein enables the virus to infect human cells.

 RNA copies of the human ADA gene can be incorporated into
the retroviral genome using a packaging cell.

Packaging cells are treated so they express:

 an RNA copy of the human ADA gene along with

o a packaging signal (P) needed for the assembly of fresh
virus particles
o inverted repeats ("R") at each end; to aid insertion of the
DNA copies into the
DNA of the target cell.
 an RNA copy of the retroviral gag, pol, and env genes but with
no packaging signal (so these genes cannot be incorporated in
fresh viral particles).
Treated with these two genomes,
the packaging cell produces a
crop of retroviruses with:

 the envelope protein needed

to infect the human target
cells
 an RNA copy of the human
ADA gene, complete with R
sequences at each end
 reverse transcriptase,
needed to make a DNA
copy of the ADA gene that
can be inserted into the
DNA of the target cell
 none of the genes (gag, pol,
env) that would enable the
virus to replicate in its new
host.

Once the virus has infected the

target cells, this RNA is reverse
transcribed into DNA and inserted into the chromosomal DNA of the
host.

What to use for target cells?

T cells

The first attempts at gene therapy for SCID children (in 1990), used
their own T cells (produced following ADA-PEG therapy) as the
target cells.

The T cells were:

 placed in tissue culture

 stimulated to proliferate (by treating them with the lymphokine,
Interleukin 2 (IL-2)
 infected with the retroviral vector
  returned, in a series of treatments, to the child

The children developed improved immune function but:

 the injections had to be repeated because T cells live for only 6–

12 months in the blood
 the children also continued to receive ADA-PEG so the actual
benefit of the gene therapy was unclear

Stem cells

Blood ("hematopoietic") stem cells:

 produce (by mitosis) all the types of blood cells, including T and
B lymphocytes
 produce (by mitosis) more stem cells, thus ensuring an
inexhaustible supply

In June of 2002, a team of Italian and Israeli doctors reported on two

young SCID patients that were treated with their own blood stem cells
that had been transformed in vitro with a retroviral vector carrying the
ADA gene. After a year, both children had fully-functioning immune
systems (T, B, and NK cells) and were able to live normal lives
without any need for treatment with ADA-PEG or immune globulin
(IG). The doctors attribute their success to first destroying some of the
bone marrow cells of their patients to "make room" for the
transformed cells.

Nine years later (August 2011) these two patients are still thriving and
have been joined by 28 other successfully-treated children most of
whom no longer need to take ADA-PEG.

Gene Therapy for X-linked SCID

Gene therapy has also succeeded for 20 baby boys who suffered from
another form of severe combined immunodeficiency called X-
linked SCID because it is caused by a mutated X-linked gene
encoding a subunit — called γc (gamma-c) — of the receptor for
several interleukins, including interleukin-7 (IL-7).
IL-7 is essential for converting blood stem cells into the progenitors
of T cells. [View]. Boys with X-linked SCID can make normal B
cells, but because B cells need T-helper cells to function, these boys
could make neither cell-mediated nor antibody-mediated immune
responses and had to live in a sterile bubble before their treatment.

Their doctors

 isolated blood stem cells from the bone marrow of each infant;
 treated the cells with a retroviral vector containing the normal
gene for the γc interleukin receptor subunit;
 returned the treated cells to each donor.

The results: Now after as long as 11 years, 19 of these boys

 are able to live normal lives at home instead of inside a sterile

"bubble";
 have normal (with some exceptions*) numbers of T cells of both
the CD4 and CD8 subsets;
 have responded to several childhood immunizations, including
diphtheria, tetanus and polio by producing both T cells and
antibodies specific for these agents.
 Antibody production is sufficiently good that most of the boys
have no need for periodic infusions of immune globulin (IG).

* Five of the little boys developed leukemia (one has died):

 in one case caused by a proliferating clone of γδ T cells in which

the vector has inserted itself in a gene (on chromosome 11)
implicated in some cases of acute lymphoblastic leukemia
(ALL).
 in a second case, the leukemia was of αβ T cells.
Gene Therapy for β-thalassemia

β-thalassemia is an inherited disease. The most severe cases result

from mutations in both copies of the gene encoding the beta chain of
hemoglobin. Many causative mutations have been identified, and
most lead to a failure to make any beta chains. The resulting
hemoglobin functions poorly and the victim requires frequent blood
transfusions.

In the 16 September 2010 issue of Nature, Cavazzana-Calvo (and

many colleagues) report a single case of successful gene therapy for
this disorder. Their patient was an 18-year old male.

Their procedure:

 Harvest blood stem cells from the patient.

 Expose them to a retroviral vector that contained
o a human gene for beta-hemoglobin complete with its
promoter, enhancer, and other control elements;
o alterations to the vector to make it safe.
 After sufficient chemotherapy to "make room" for them, the
patient was injected with these cells.

The result: Almost three years later, the patient is well and no longer
requires periodic blood transfusions. One-third of his hemoglobin is
now manufactured by the red-cell precursors descended from the
gene-altered stem cells.

Adenovirus Vectors

Adenoviruses are human pathogens responsible for some cases of the

human "cold". Modified versions of two strains are currently being
used as vectors in gene therapy trials.

Advantages:

 Unlike retroviral vectors, they do not integrate into the host

genome and thus should not be able to disrupt host genes (It was
 such disruption that caused some X-linked SCID patients treated
with a retroviral vector to develop leukemia).
 They can infect nondividing cells with high efficiency.

Disadvantages:

 They elicit a powerful immune response, both by T cells and by

B cells (antibodies) so repeated doses soon lose their
effectiveness.
 Many people already have antibodies against the virus from
earlier "colds", and these can inactivate the vector at the outset.
A recent trial of an HIV vaccine using an adenovirus as the
vector was halted when it was found not only not to be effective
but, in people with preexisting high levels of anti-adenovirus
antibodies, may have even increased their susceptibility to HIV.