1 | P a g e

Adenosine deaminase (ADA)

Gene Therapy

2 | P a g e

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

3 | P a g e

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.

4 | P a g e

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

5 | P a g e

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

6 | P a g e

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.

7 | P a g e

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

8 | P a g e

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.

9 | P a g e

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.

10 | P a g e

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.

11 | P a g e

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.

12 | P a g e

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.

13 | P a g e

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

14 | P a g e

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

15 | P a g e

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

16 | P a g e

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

17 | P a g e

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.

18 | P a g e

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.

19 | P a g e

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.