Adenosine deaminase (ADA) Gene Therapy

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Adenosine deaminase (ADA)
Gene Therapy

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Contents
 Introduction
 Genetics
 ADA-scid
 Immune Defects
 Non-Immune Defects
 Treatment
 Hematopoietic Stem Cell Transplantation
 Enzyme Replacement Therapy with PEG-ADA
 Gene therapy for ADA-SCID
 Pre-clinical studies of gene therapy for
ADA-SCID
 Gene transfer to Hematopoietic stem cells (HSCs):
 Gene transfer to T lymphocytes
 Clinical trials of gene therapy for ADA
deficiency
 T cell gene therapy
 T cell depleted bone marrow and peripheral blood lymphocyte gene
therapy
 CD34+ cells gene therapy
 Conclusion
 References

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Adenosine deaminase
Adenosine deaminase (also known as adenosine aminhydrolase or ADA) is an enzyme
involved in purine metabolism. It is needed for the breakdown of adenosine from food
and for the turnover of nucleic acids in tissues.
Present in virtually all mammalian cells, its primary function in humans is the
development and maintenance of the immune system. However, the full physiological
role of ADA is not yet completely understood.
ADA deficiency may be present in infancy, childhood, adolescense, or adulthood. Age
of onset and severity is related to some 29 known genotypes associated with the disorder.
As an enzyme of the purine salvage pathway, adenosine deaminase (ADA) catalyzes the
deamination of adenosine and 2′-deoxyadenosine, as well as several naturally occurring
methylated adenosine compounds. The deamination of adenosine and 2′-deoxyadenosine
gives rise to inosine and deoxyinosine, respectively. Further conversion of these
deaminated nucleosides leads to hypoxanthine, which can be either transformed
irreversibly into uric acid or salvaged into mononucleosides.
Although ADA is present in all cell types, its enzyme activity differs considerably
among tissues. The highest amounts in humans are found in lymphoid tissues,
particularly the thymus, the brain, and gastrointestinal tract. The ADA enzyme is
ubiquitously expressed both intracellularly and on the cell surface where it complexes
with two molecules of CD26 as a combined protein.

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Figure . The adenosine deaminase (ADA) metabolism. ADA is an enzyme of the purine salvage pathway,
which catalyzes the irreversible deamination of adenosine and 2′-deoxyadenosine into inosine and 2′-
deoxyinosine, respectively. Most adenosine derives from endogenous breakdown of ATP and
degradation of RNA, or is taken up exogenously by ubiquitously expressed nucleoside transporters.
Unlike adenosine, 2′-deoxyadenosine is formed by DNA degradation is predominantly catabolized by
ADA. Further conversion of inosine nucleoside leads to hypoxanthine, which can either enter a non-
reversible pathway to uric acid or salvaged back into other mononucleosides. In the absence of ADA, the
presence of these alternatives “bypasses” pathways results in normal concentrations of the catabolic
products of the enzyme reaction in patients with ADA-SCID. Conversely, the levels of ADA substrates,
adenosine and 2′-deoxyadenosine, are not only found in increased amounts in extracellular body fluids,
but they also “spill over” into additional pathways normally only minimally utilized, thus contributing to
the pathogenic mechanisms of the disease.
Genetics
The enzyme adenosine deaminase is encoded by a gene on chromosome 20. ADA
deficiency is inherited in an autosomal recessive manner. This means the defective gene
responsible for the disorder is located on an autosome (chromosome 20 is an autosome),
and two copies of the defective gene (one inherited from each parent) are required in
order to be born with the disorder. The parents of an individual with an autosomal

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recessive disorder both carry one copy of the defective gene, but usually do not
experience any signs or symptoms of the disorder.
Figure: Adenosine deaminase deficiency has an autosomal recessive pattern of inheritance.
ADA-SCID
Adenosine deaminase deficiency is the second-most prevalent form (approximately
20%) of SCID. The overall incidence in Europe is estimated to range between 1:375,000
and 1:660,000 live births. ADA-deficient patients suffer from lymphopenia, severely
impaired cellular and humoral immune function, failure to thrive, and a rapidly fatal
course due to infection. Moreover, autoimmune manifestations are commonly observed

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in milder forms of the disease. Currently available therapeutic options include bone
marrow transplantation (BMT), enzyme replacement therapy with bovine ADA (PEG-
ADA), or hematopoietic stem cell gene therapy (HSC-GT).
Immune Defects
Lymphopenia and attrition of immune function over time are the two findings
common to all presentations of ADA deficiency. It is associated with thymic hypoplasia
and a severe depletion of all three major categories of lymphocytes, T-, B-, and NK-cells.
Absence of cellular and humoral immunity and a rapidly fatal course due to infections
with fungal, viral, and opportunistic agents are characteristic of early onset forms of
ADA deficiency. Total immunoglobulin levels may be only slightly depressed at birth
due to the maternal contribution of IgG, whereas both IgM and IgA, which ordinarily do
not cross the placental barrier, are often absent. However, once IgG levels decline as
maternal antibodies are cleared, a pronounced hypogammaglobulinemia signals the
absence of humoral immunit.
About 20% of ADA-SCID cases occur later in childhood (delayed) or beyond
(late/adult onset). Delayed or late-onset patients have significant immunodeficiency, but
variable clinical manifestations. These forms show progressive immunological and
clinical deterioration, often associated with autoimmune manifestations, including
hemolytic anemia, and immune thrombocytopenia. Serum immunoglobulin levels are
altered in late-onset patients, with IgG2 levels being highly reduced or absent. IgE levels
are elevated and often associated to eczema and asthma. An inability to produce
antibodies against polysaccharide and pneumococcal antigens was frequently found in
ADA-SCID patients with milder forms of the disease.

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Non-Immune Defects
The initial and most devastating presentation of ADA-SCID is due to the immune
defects. Nonetheless, several non-immune abnormalities have been described in ADA
deficiency, indicating that this disease should be considered a systemic metabolic
disorder. ADA is ubiquitously expressed in all cell types; when absent, the systemic
metabolic toxicity is frequently associated with organ damage. These include:
 Hepatic and renal disease
 Skeletal alterations
 Neurological abnormalities
 Behavioral impairments
Because complications from infections usually predominate in the clinical presentation
of infants with ADA deficiency, the full spectrum of non-immunologic manifestations
and their natural course may be obscured. It is important to note, that several
abnormalities have been described in few patients only, and might reflect effects of
infectious agents rather than primary defects due to ADA deficiency: i.e., renal and
adrenal abnormalities, phyloric stenosis, and hepatic disease.
Treatment
Treatments include:
 bone marrow transplant
 gene therapy
 ADA enzyme in PEG vehicle

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Figure . Current therapeutic options in ADA-SCID and reported autoimmune manifestations after
treatment. Immune reconstitution in ADA deficiency can be achieved by bone marrow
transplantation, enzyme replacement, or gene therapy; nonetheless recovery of immune functions
may vary depending on the applied treatment and patient’s characteristics. Treatment of choice
remains bone marrow transplantation from an HLA-identical sibling donor, while transplants from
alternative donors are associated with high morbidity and mortality. Enzyme replacement therapy
using pegylated bovine ADA is a non-curative treatment requiring weekly intramuscular injections
with PEG-ADA. ADA-SCID has been the pioneer disease for the development of human gene
therapy. It is based on the reinfusion of autologous HSC transduced with a retroviral vector
containing the ADA cDNA. Variable degrees of immune reconstitution can be achieved by these
treatments, but onset of autoimmunity is of concern in post-treatment ADA-SCID patients. Reported
autoimmune manifestations include: autoimmune hypothyroidism, diabetes mellitus,
thrombocytopenia, hemolytic anemia, and development of anti-ADA antibodies. HLA, human
leukocyte antigen; BMT, bone marrow transplantation; MUD, matched unrelated donor; PEG-ADA,
pegylated bovine ADA; HSC, hematopoietic stem cell; PSC, pluripotent stem cell; CLP, committed
lymphocyte precursor; NK, natural killer cell.

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1.Hematopoietic Stem Cell
Transplantation
The treatment of choice for ADA-SCID is bone marrow transplantation (BMT). When
a matched donor is available, the success rate of BMT can be as high as 90%.However,
mortality and morbidity increase dramatically when transplantation is performed from a
mismatched donor.
Hematopoietic stem cell-transplantation (BMT) from allogeneic human leukocyte
antigen (HLA)-compatible sibling donors resulting in long-term survival and effective
immune reconstitution is the treatment of choice for patients with ADA-SCID and other
severe variants of primary immunodeficiencies. Since less than 20% of ADA-SCID
patients have access to HLA-matched family donors, transplants are often performed
from mismatched family or matched unrelated donors. A recent retrospective analysis on
the specific outcome of transplants for ADA-SCID collected data from several
multicenter studies and analyzed the survival of 106 patients who received a total of 119
transplants. BMT from matched sibling and family donors had a significantly better
overall survival (86 and 81%) in comparison to BMT from matched unrelated (66%) and
haploidentical donors (43%). Indicating that despite recent progress in transplantation,
the use of alternative donors is still associated with a reduced overall survival. This is
further complicated by the fact that ADA-SCID patients are more difficult to transplant
especially from unrelated and haploidentical donors possibly due to their need for
conditioning and the underlying metabolic nature of the disease.
In summary, the results obtained with transplantation from HLA-identical siblings or
family donors indicate superior donor/host compatibility and outcome both in terms of
survival and sustained immune recovery. Whereas the current evidence suggests that
haploidentical donor transplants (performed with or without conditioning) have a poor
chance of success and are therefore only undertaken if no other treatment options are
available.

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2.Enzyme Replacement Therapy with PEG-
ADA
Enzyme replacement therapy with PEG-ADA was developed as lifesaving, not
curative treatment for patients lacking an HLA-compatible donor. Attachment of PEG
through lysine residues confers several therapeutically beneficial properties to ADA. This
chemical modification of the bovine enzyme reduces its immunogenicity and prevents its
degradation by plasmatic proteases as well as the binding of neutralizing antibodies.
Thereby the circulating life of the compound is prolonged from minutes to days as
clearance from the circulation is inhibited. Cellular uptake of PEG-ADA is insignificant
and its distribution is limited to the plasma. Enzymatically active ADA continuously
circulates and eliminates accumulating adenosine and 2′-deoxyadenosine metabolites.
The principle of exogenous PEG-ADA administration is based on the direct conversion
of accumulating ADA substrates in the plasma and the indirect reduction of intracellular
toxic metabolites by diffusion.
To date more than 150 patients worldwide have received this treatment. PEG-ADA is
usually administered weekly or bi-weekly by intramuscular injections throughout life. In
general, PEG-ADA treatment seems to be well tolerated, with clinical benefits
appreciable after the first month of therapy. Studies have shown that upon the initiation
of PEG-ADA therapy, the absolute numbers of circulating T- and B-lymphocytes and
NK-cells increase and protective immune function develops. Although only limited
information is available, some analysis indicated that about half of PEG-ADA treated
patients discontinued IVIg, whereas long-term follow-up suggests that immune recovery
is often incomplete. Two retrospective studies showed that despite initial improvements,
the lymphocyte counts of all PEG-ADA treated patients were below the normal range at
all times. A gradual decline of mitogenic proliferative responses occurred after a few
years of treatment and normal antigenic responses occurred less than expected. No toxic
or hypersensitivity reactions have been reported with PEG-ADA administration.

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However, several other side effects have been reported including manifestations of
immune dysregulations including autoimmunity (type I diabetes, hypothyroidism,
immune thrombocytopenia, hemolytic anemia) and allergic manifestations. An additional
concern with PEG-ADA beyond about 8–10 years is the emergence of serious
complications, including lymphoid and hepatic malignancies, and progression of chronic
pulmonary insufficiency.
Figure 3. Immune reconstitution and development of autoimmunity after PEG-ADA treatment. Enzyme
replacement therapy with pegylated bovine ADA is a lifesaving but non-curative treatment for ADA-
SCID patients. It provides metabolic detoxification and protective immune function with patients
remaining clinically well, but immune reconstitution is often suboptimal and may not be long-lived.
Shortly after initiation of PEG-ADA treatment, patients show recovery of B-cell counts, followed by a
gradual increase in T-cell numbers and reconstitution of immune cell functions. However, the long-
term consequences of PEG-ADA treatment are unknown. Immune recovery in B and T- cells is below
normal levels. Major concerns are the susceptibility to opportunistic infections and the development of
autoimmunity due to lymphopenia with gradual decline of immune functions and perturbation of T-
and B-cell tolerance.

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The main side effect associated with the use of PEG-ADA is the development of anti-
ADA antibody. The development of specific IgG antibody to bovine peptide epitopes of
PEG-ADA has been reported by several groups and often coincides with an improvement
in humoral immunity. In about 10% of treated patients, inhibitory antibodies lead to the
enhanced clearance of PEG-ADA with subsequent decline in metabolic parameters and
immune function.
3.Gene therapy for ADA-SCID
Since the ADA gene was cloned, ADA-SCID has been regarded as the perfect model
disease for gene therapy. This is firstly due to the fact that the target tissue and the cells
are well defined. ADA is expressed in all body tissues but the pathology is largely due to
the damage to the immune system, particularly the T cells. These cells and their
precursors in the hematopoietic lineage are easily accessible, can be genetically
engineered to produce ADA ex vivo and reinfused into the patient. Moreover, based on
results obtained from ADA-deficient patients treated by BMT, the genetically modified
cells reintroduced to the patient are expected to have a selective advantage over the
unmodified defective cells.
Another important consideration is the fact that the ADA complementary DNA
(cDNA) to be delivered is relatively small (1.1kb) and therefore can be accommodated in
virtually all gene therapy delivery does not need to be regulated by a cell-selective
promoter and therefore, well characterized viral promoters driving high levels of
constitutive expression can be used. In addition, studies performed on heterozygote
carriers have shown that individuals with as little as 10% of normal ADA activity have
no abnormality of immune function. Thus, on the basis of these observations, the
treatment of a fraction of the cell population may be enough to provide improvement in
the condition.

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Pre clinical studies of gene therapy for ADA-
SCID
The development of a new therapy follows a succession of steps, from in vitro
experiments, to in vivo studies on animal models and, if successful, to clinical trials.
Nearly all the studies on ADA-SCID gene therapy have used different versions of
retroviral vectors as gene delivery systems. The main advantage of this system is that the
transgene (in this case the ADA cDNA) will integrate into the genome of the recipient
cell and therefore should be expressed as long as the transduced cell is alive. Two main
cellular targets have been used:
1. Hematopoietic stem cells (HSCs)
2. T lymphocytes
I. Gene transfer to Hematopoietic stem cells (HSCs):
The obvious target cells for gene therapy for ADA-SCID as well as for other
conditions curable with BMT are the HSCs. Ideally, infection of these cells with a
retrovirus containing the therapeutic transgene would result in the integration of this
transgene in these progenitor cells, leading to the repopulation of all hematopoietic
lineages with the genetically modified cells. Moreover, this approach would be a
once-only procedure.
The initial studies involved transduction of murine marrow cells ex vivo under a
variety of culture and infection conditions, followed by transplantation of these
transduced cells into irradiated recipient mice. Many studies showed successful
transfer as well as long term-term in vivo expression of different trangenes including
the ADA cDNA. For example, a particular study described the expression of the
human ADA in all hematopoietic lineages of primary recepients 4 months after
transplantation as well as expression of this transgene in the peripheral blood of

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secondary recepients. All together, these studies demonstrated the stability of transfer
and expression of these murine progenitor cells. In an attempt to create an animal
model of the disease, an ADA knockout mouse has also been generated but the
phenotype of this mouse is very different from the human condition. In particular, no
signs of immunodeficiency could be detected in these animals.
Following the success obtained with murine models, the next step was to perform the
same type of experiment on larger animals (cats, dogs), including non human primates
(rhesus monkey). Coculture of early progenitors from the marrows of these animals with
a retroviral packaging cell lineage subsequent autologous transplantation of retrovirally
transduced cell resulted in a multi-lineage genetic modification that lasted more than two
after the gene transfer. However, in all the experiments the level of expression of the
human ADA transgene was significantly lower than that achieved in the murine models
.in some cases, the trangene stopped being expressed after 3 to 4 months.
As far as safety is concerned, no noticeable side-effects related the gene transfer
procedure were observed when retroviral vectors were derived cell lines that were free
from helper virus. However, lymphomas occurred in immune suppressed monkeys
exposed to murine amphoteric murine helper virus, stressing the importance of using
helper free virus producing cell lines.
The final step in these preclinical studies of gene transfer to HSCs was to transducer
human HSCs. This process required the isolation of human HSCs which were as
primitive and totipotent as possible and optimization of the condition to transducer these
cells. Long-term bone marrow culture, an approach that had been successfully used for
autologous BMT , was assayed. The results showed a high frequency of gene transfer of
retro viruses. Another approach involved the isolation of cells from bone marrow on the
basis of their expression of the CD34 molecules. CD34+ consist of the earliest
hematopoietic progenitors and are thought to contain HSCs. in vitro studies demonstrated
that these CD34+ cells could be infected by retroviruses , although at a lower efficiency

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than that achieved with long-term bone marrow culture. An alternative source of HSCs
was found in the human umbilical cord. This blood is rich in progenitor cells and cord
blood transfusion had already been used for a variety of hematopoietic diseases.
Moreover, progenitors and CD34+ cells from human umbilical cord blood can be
transduced by retroviruses at frequency similar or greater than bone marrow-derived
cells.
II. Gene transfer to T lymphocytes
The technical difficulties associated with the isolation, culture and transduction of
HSCs led certain groups to target T lymphocytes for the correction of the ADA
deficiency. Primary cultures of T lymphocytes from patients were retrovirally transduced
and these ADA-expressing cells were shown to grow for a significantly longer period in
vitro, compared to the untransduced cells. This observation confirmed the assumption
that ADA transduced cells had a survival advantage. These results were extended in vivo
by injection of ADA-transduced peripheral blood lymphocytes from ADA-SCID patients
into immunodeficient mice. Only the ADA-transduced cells were able to show long-
term survival, accompanied by immunoglobin production and development of antigen-
specific T cells. Although, these results suggested that t-cell modification could
reconstitute a certain degree of immune function.
Clinical trials of gene therapy for ADA
deficiency
T cell gene therapy
The first clinical trial of gene therapy for ADA was started on two girls in the USA in
1990. Both were on PEG-ADA therapy and had shown a good initial response to this
treatment, followed by a deterioration of the lymphocyte number and response. The gene
therapy protocol involved infection of peripheral T cells from the patients with a

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retrovirus containing human ADA cDNA in a combination with recombinant human
interleukin 2 (rIL-2) and an anti-CD3 antibody which both stimulated T-cell
proliferation and thus improved retroviral transduction. The expanded T-cell population
was then returned to the patients 9 to 12days later. The procedure was repeated 12 ties at
regular intervals for each patient over a period of 18-24 months. From one round of
transduction to the next, the efficiency of transduction varied from 0.1 to
10%.polymerase chain reaction analysis of peripheral cells performed up to 2 years after
the last infusion showed that the retroviral sequence were present at a rate 0.3 copy per
cell in patient 1 while the level of transduced cells was only 0.1 to 1% in patient 2.
Interestingly, these levels were stable over 2 years, a time period far longer than
expected.
The level of enzymatic ADA activity followed the results obtained by PCR, i.e. a
significant rise in patient 1 but no change in patient 2. In both patients, the T-cells count
rose rapidly after treatment and stabilized in the normal range for patient 1 and with a
slight increase in patients 2. For both patients, cell-mediated immunity, T-cell immune
response in vitro and humoral immune functions improved significantly. However, the
continuous administration of PEG-ADA complicated the outcome of this trial. At
present, the dose of PEG-ADA is being reduced.
T cell depleted bone marrow and peripheral blood
lymphocyte gene therapy
Another trial was performed on two patients with similar clinical conditions: the
patients were treated with PEG-ADA and gene therapy was started when this treatment
failed to have any effect on immunological parameters. The trial involved infusion of
transduced T cells into patients, with increasing numbers of transduced HSCs later on.
The two cell populations were transduced with slightly different retroviral vectors this
allowed an easy and precise evaluation of the efficiency of both approaches. Both
patients received infusions of gene-modified peripheral blood lymphocytes and HSCs

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over a 2-year period. Initially, all vector positive cells were derived from transduced
peripheral blood lymphocytes. However, 1 year after the end of the treatment, the
lymphocytes analyzed showed a bone marrow origin.
Despite the presence of these transduced cells, the level of ADA enzymatic activity
remained low(less than 20% of the normal values). The immune reconstitution appeared
to be more consistent than in trial with an increase in the absolute number of
lymphocytes as well as in the number of active T cells, in both children.
CD34+ cells gene therapy
Retroviral-mediated gene transfer to bone marrow CD34+ Cells was attempted on
three ADA-deficient children in a once-only procedure. The gene transfer resulted in 5-
12% of the cells being transfected in vitro. Transduced cells were detected 3 months
after treatment (6 months after treatment for one patient), but no ADA gene expression
was detected at any time.
In another trial umbilical cord blood was the source of CD34+ cells. Three infants
were diagnosed prenatally and treated by autologous transplantation of retrovirally
transduced CD34+ cells. For all three patients PEG-ADA treatment started a few days
after birth. Four years later, the number of gene- containing T lymphocytes has reached
1-10% whereas the frequency of other hematopoietic and lymphoid cells remained
below 0.1%. However, cessation of the PEG-ADA treatment led to a decline in immune
function despite the presence of ADA positive T lymphocytes.
Conclusion
Despite the initial optimism, no trial has yet achieved the objective of clinical cure.
Very few patients have been treated and long-term benefits of ADA-SCID on immune
function remain to be clearly demonstrated. Moreover, the concomitant administration
of PEG-ADA complicates the issue.

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However, a number of encouraging points have been observed. It has been
demonstrated that the transduction of HSCs is possible and that the T lymphocytes
generated from these progenitors remain in the circulation for much longer period than
initially predicted. Importantly, no side-effect was reported as a result of gene transfer.
The most significant concern was the contamination of the ADA-encoding retroviruses
with wild type replication-competent retroviruses. But all the assays performed to detect
wild type viruses were negative. Another safety aspect was the potential for insertional
mutagenesis due to random integration of the retrovirus-derived transgene.
The initial studies have underlined the feasibility of gene therapy and have
highlighted the problems that need to be overcome. Furthermore, they have given further
insight into the biology of the hematopoietic cell lineage that may lead to improve
second generation ADA-SCID gene therapy trial protocols.

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References
1. Abuchowski, A., van Es, T., Palczuk, N. C., and Davis, F. F. (1977). Alteration of
immunological properties of bovine serum albumin by covalent attachment of
polyethylene glycol. J. Biol. Chem. 252, 3578–3581.
2. Aiuti, A. (2004). Gene therapy for adenosine-deaminase-deficient severe
combined immunodeficiency. Best Pract. Res. Clin. Haematol. 17, 505–516.
3. Aiuti, A., Ficara, F., Cattaneo, F., Bordignon, C., and Roncarolo, M. G. (2003).
Gene therapy for adenosine deaminase deficiency. Curr. Opin. Allergy Clin.
Immunol. 3, 461–466.
4. Aiuti, A., Cassani, B., Andolfi, G., Mirolo, M., Biasco, L., Recchia, A., Urbinati,
F., Valacca, C., Scaramuzza, S., Aker, M., Slavin, S., Cazzola, M., Sartori, D.,
Ambrosi, A., Di Serio, C., Roncarolo, M. G., Mavilio, F., and Bordignon, C.
(2007). Multilineage hematopoietic reconstitution without clonal selection in
ADA-SCID patients treated with stem cell gene therapy. J. Clin. Invest. 117,
2233–2240.
5. Aldrich, M. B., Chen, W., Blackburn, M. R., Martinez-Valdez, H., Datta, S. K.,
and Kellems, R. E. (2003). Impaired germinal center maturation in adenosine
deaminase deficiency. J. Immunol. 171, 5562–5570.