TPI Research Project Progress at Kings College Hospital, London

The TPI Research Team

From left to right: Dr. Art Ationu, Dr. Anne Humphries and Dr. Roopen Arya.

Select from research project progress pages below:-

Latest TPI research articles published by the Dr. Mark Layton Kings College and Professor Susan Hollan, Hungary team Include:-

Published During 1999

Published During 1998

Published During 1997

Published During 1996

Published During 1995

Triosephosphate Isomerase Enzyme Replacement Therapy Research Progress Report (1997-1998).


Triosephosphate isomerase (TPI) is an ubiquitously expressed enzyme that catalyses the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate in the energy-generating glycolytic pathway. Homozygous TPI deficiency is characterised biochemically by markedly reduced enzyme activity in all tissues accompanied by metabolic block in glycolysis with accumulation of intracellular DHAP particularly in red cells. Clinical TPI deficiency is a rare autosomal recessive disorder (only 30 cases world-wide have been reported to date) characterised by chronic haemolytic anaemia, recurrent infections, cardiomyopathy, severe and progressive neuromuscular disease. These complications are frequently life-threatening. Although first reported over 30 years ago, TPI deficiency remains without effective therapy. The possibility of enzyme replacement therapy has not hitherto been studied.

Our current research efforts have been aimed at examining metabolic correction of TPI deficiency in vitro by exogenous administration of functional enzyme or cocultivation of deficient cells with normal donor cells, as a first step toward clinical therapy.

In vitro studies

Culture of deficient cells with exogenously administered TPI.Primary skeletal muscle and lymphoblastoid cells derived from TPI deficient patients provide a model system for the disease as they are directly relevant to the clinical manifestations of the disorder, and exhibit the biochemical characteristics of TPI deficiency. These deficient cells were cultured in the presence of exogenous enzyme. The experimental approach utilised in our in vitro studies establishes the proof of principle that a) transfer of active enzyme to deficient cells is feasible and b) the DHAP metabolic block in TPI deficiency is reversible. Intracellular enzyme activity increased in deficient muscle and lymphoblastoid cells when cultured in the presence of normal human plasma, or purified rabbit muscle TPI as source of functional TPI. Metabolic correction by exogenously administered TPI was not however sustained due to depletion of functional enzyme in the culture medium. This suggests frequent or continuous therapy with TPI may be necessary for sustained effect. Significantly, metabolic correction of the defect in TPI deficiency was successfully achieved in cells from all three patients studied, irrespective of the genetic alteration identified by molecular analysis as responsible for TPI deficiency.

Coculture of deficient cells with normal donor cells
Skeletal muscle impairment and progressive neurological dysfunction are hallmarks of TPI deficiency. The prospect of effective therapy for TPI deficiency may be limited by the need to replace defective TPI enzyme in tissues worst affected by the disorder, such as neural and muscle cells.

Bone marrow transplantation (BMT) has been used to clinical benefit with partial correction of the neurological manifestation in some lysosomal storage disorders. The principle underlying use of BMT as a therapeutic option in metabolic disorders is the secretion and recapture of functional enzyme by donor and host cells respectively. This may be tested in vitro in a coculture system.

To establish whether sustained correction of the metabolic defect in TPI deficient cells was possible, deficient primary skeletal muscle and lymphoblastoid cells were incubated in the presence of normal donor cells in an in vitro coculture model system in which TPI deficient and normal cells are separated by a semi-permeable membrane. Results clearly show increased intracellular TPI activity with a reduction in elevated DHAP to normal level in deficient cells cocultured with normal cells as source of functional enzyme. We also observed a reversal of metabolic block in TPI deficient bone marrow colony forming unit (CFU) cells cocultured with normal leukocytes from an HLA-identical related donor in a feeder layer culture system for 7 days, a finding that confirms the possibility of sustained metabolic correction.

In summary, results from in vitro studies suggest that it is possible to correct the primary metabolic defect in TPI deficiency in cells directly relevant to the clinical manifestations of the disorder when cultured in the presence of exogenously administered TPI or cocultured with normal cells as source of functional enzyme. Replacement of defective TPI enzyme with exogenous functional enzyme resulted in a reduction in DHAP to normal levels in deficient cells. While this provides a rationale for enzyme replacement therapy in TPI deficiency, reversal of metabolic block by exogenous TPI was transient, implying continuous enzyme therapy will be necessary for durable benefit. To address this question, an in vitro coculture model was used to confirm the feasibility of sustained correction in TPI deficiency. Collectively, these data have important implications for the potential role of allogeneic BMT and gene therapy as approaches to sustained delivery of TPI to neural and muscle cells in vivo.

In vivo studies

Red cell transfusion therapy results in clinical improvement in adenosine deaminase (ADA) and nucleoside phosphorylase (PNP) deficiencies. Evidence from our in vitro studies suggests that it is possible to correct the metabolic defects of TPI deficiency. These in vitro studies led us to propose that functional enzyme administered to deficient patients might lead to reversal of the metabolic defects in vivo. In the absence of human enzyme for clinical use the feasibility of replacement by red cell transfusion therapy has been evaluated in one patient with homozygous TPI deficiency. Extrapolation of our in vitro data to the clinical treatment of TPI deficiency will require rigorous analysis. Preliminary results have demonstrated changes in both TPI and DHAP levels after transfusion which persist for at least 2 weeks. This will be validated in further studies after exchange transfusion to be undertaken at Kingís College Hospital in May 1998.

Future studies

1. We have demonstrated uptake of TPI in normal human skeletal muscle and brain cell lines, as well as lymphoblastoid cells after culture in the presence of exogenous enzyme. These observations suggest the existence of a transport mechanism that permits transfer of TPI across the cell membrane. The mechanism of uptake of TPI remains to be determined. If understood this might allow therapy to be targeted via receptors on lesional cells.

2. Despite evidence for reversal of the metabolic defect in TPI deficient muscle and lymphoblastoid cells, it is unclear whether this approach would prove effective in correction of the neurological effects of TPI deficiency. Future studies to define the feasibility of enzyme replacement strategies in neural cells are warranted. In particular, efforts should be directed towards testing metabolic correction of TPI deficiency in coculture studies involving neural cells. This is a key question in the context of the possible role of BMT. The efficacy of BMT in TPI deficiency has not been evaluated and it remains unclear whether it will arrest or reverse the prominent neurological effects of the disorder.

3. Efficient delivery of active enzyme will be essential to the treatment of TPI deficiency. Further in vitro studies are required to define the dosage of enzyme necessary to achieve functional restoration of glycolysis. For treatment to be successful, an active form of enzyme suitable for clinical use needs to be developed. Developing a genetically engineered human enzyme would require major biotechnology support. Purified bovine (cow) enzymes are already in clinical use for the treatment of ADA deficiency. Cloning of the gene for bovine TPI would enable comparison against the primary structure of human TPI and degree of homology determined. In turn this will give an indication of whether immunological rejection is likely to be a significant problem if bovine enzyme is used clinically. This would complement our research groupís ongoing collaboration with St. Georgeís Hospital Medical School aimed at establishing the feasibility of packaging functional enzyme in red cells for clinical use.


1. Metabolic correction of triosephosphate isomerase deficiency in vitro by complementation.
Biochem. Biophys. Res. Commun. (1997); 232:528.

2. Prenatal diagnosis of triosephosphate isomerase deficiency due to codon 104 mutation.
Blood (1996); 87: 4507.

3. Evidence for founder effect of the Glu 104 Asp substitution and identification of new mutations in triosephosphate isomerase deficiency.
Human Mutation (1997); 10:290.

4. Prospect for enzyme replacement therapy in a red cell enzymopathy: Triosephosphate isomerase deficiency.
Blood (1998); (submitted).

5. Feasibility of fresh frozen plasma as a replacement therapy for human triosephosphate isomerase deficiency.
Clin. Sci. (1997); 92:12p (presented at the Medical Research Society meeting, London 1996).

6. Sodium butyrate upregulates triosephosphate isomerase gene expression in human endothelial cell line.
Clin. Sci. (1997); 92:6p (presented at the Medical Research Society meeting, London 1996).

7. Metabolic correction of triosephosphate isomerase deficiency in vitro by complementation.
Br. J. Haematol. (1997); 97 (Suppl 1):73 (presented at the British Society of Haematology Meeting, Harrogate 1997).

8. Prospect for enzyme replacement therapy in a red cell enzymopathy: Triosephosphate isomerase deficiency.
Br. J. Haematol. (1998); 101 (Suppl. 1):51 (presented at the British Society of Haematology Meeting, Glasgow 1998).

London Meeting January 7th 1998 at Kings:

The TPI progress meeting took place at Kings College Hospital, London on January 7th 1998. Professor Susan Hollan from Hungary, Dr. Mark Layton and Dr. Roopen Arya from Kings, the 2 Kings TPI Research Scientists Art Ationu and Anne Humphries and James Stewardson's parents attended.

The main points from the meeting were as follows:-

  • The positive results from the analysis undertaken after James's December 12th transfusions were discussed in detail. The same analysis needs to be repeated after James undergoes his 5th transfusion in February. Although the blood given to James was meant to be free of white cells, there is a very small possibility there were white cells present in the transfused blood, which could cloud, slightly, the very positive results. For more details see Treatment page.
  • The decision regarding James undergoing a bone marrow transplant will probably be made after analysis of the February transfusion.
  • A meeting is taking place with a team from Kings and another research establishment in London to discuss the possibility of developing a human form of TPI. Currently this team are working with a manufactured synthetic form of rabbit TPI.
  • The TPI research work completed by Kings since the programs inception in December 1995 were discussed. The work has included:

    - successfully correcting the metabolic block in donor cells and then James's cells from red cells in vitro
    - the plasma therapy work, which later proved unsuccessful when attempted on James
    - James's muscle biopsey tests proved the weakness involved the nerves rather than the muscles

    Kings will issue a progress status report within the next few weeks.

  • Susan Hollan discussed her research in detail, her team have discovered some differences in the two Hungarian TPI deficient brothers, one of which has the neurological symptoms the other has not. It was agreed to send blood samples of James to Hungary and possibly other TPI children, so Susan can repeat her work on James's cells. It was also agreed that the james Stewardson TPI Trust would further fund Professor Susan Hollans research for another year.

    For further information regarding TPI research you can e-mail Dr Roopen Arya at:

    Send E-Mail to Dr Roopen Arya at Kings College Hospital, London