HUMAN PROOF-OF-PRINCIPLE STUDY IN SEVERE APLASTIC / HYPOPLASTIC ANAEMIA

 Aplastic/Hypoplastic Anaemia

Aplastic Anaemia is defined as the failure of bone marrow to produce blood cell components. Diagnosis is confirmed by a complete blood cell count (CBC), which reveals varying degrees of anaemia - mild, moderate, or severe on the basis of the severity of the pancytopenia. Aplastic anaemia is a disease with an incidence of 0.6 to 6 cases per million population and up to 14 cases per million population in the US and Far East respectively. Current available treatments include immunosuppression, typically with horse antithymocyte globulin and cyclosporine, and allogeneic bone marrow or peripheral blood (G-CSF) stem cell transplantation from an HLA-matched donor.

Information Reference Website:

Leukaemia Research Fund click here>>

(http://www.lrf.org.uk/en/1/infdispatapl.html)

Retrodifferentiation - A treatment option for severe aplastic/hypoplastic anaemia

The potential use of autologous retrodifferentiated pluripotent stem cells for the treatment of aplastic/hypoplastic anaemia is an extremely attractive option when compared to the conventional therapies above. This is due to the following reasons:

• Graft versus host disease (GVDH) and associated infections are precluded due to the autologous approach

• A wider application potential of the retrodifferentiation technology is enabled, particularly for those individuals awaiting a stem cell transplant from an unrelated donor, or those for which there is no matched sibling

• The procedure is relatively less invasive, rapid, reproducible and generates up to 2.7 billion pluripotent cells per litre of blood (cf. 72 million cells per litre in 5 to 6 days with peripheral blood extraction using expensive and harmful growth factors)

• Economically, it is far more affordable to developing countries when compared to expensive immunosuppression or conventional stem cell transplant therapies

• Alloimmunisation due to repeated transfusions with platelets and packed red blood is totally abrogated.

Human Clinical Study – Retrodifferentiated Stem Cells

A four-patient human proof-of-principle clinical study applying the core Retrodifferentiation in aplastic/hypoplastic anaemia patients (3 severe aplastic anaemia, 1 hypoplastic anaemia) has been completed according to established endpoint criteria⁽¹⁾. This collaborative study between the Indian Council for Medical Research and GG HaemHealth Technologies (Pvt) Limited, and in consultation with TriStem Corporation Limited, utilised an autologous stem cell transplant/semi-automated approach.

Patients were apheresed by processing 2-3 times their body volume to collect buffy coats and white blood cells; the latter were then subjected to the retrodifferentiation process for 2 hours and 30 minutes in an enclosed system. The retrodifferentiated stem cells were washed and subsequently infused into the patient (Fig 1) and from there on, the patients were monitored for any side effects. Clinical tests were performed before and after infusion for the duration of the clinical study. All patients continue to be monitored for up to 2 years.

Washing of 2 hr and 30 minutes Retrodifferentiated Stem Cells

Infusion of Retrodifferentiated Stem Cells

Four patients have been treated with the proprietary retrodifferentiation technology without conditioning regimen such as chemotherapy or immunosuppressive drugs, and all four have successfully tolerated the retrodifferentiated pluripotent stem cells. No side or adverse effects have been observed and the patients have shown positive signs of physical recovery.

The overall clinical findings in these patients are as follows:

Engraftment and repopulation of the bone marrow (fig 2) – leading to a significant sustained increase in neutrophil and platelets count above 1,000 and 20,000 in peripheral blood respectively as measured from the second day of infusion; an unprecedented clinical outcome when compared to conventional stem cell protocols. Fourteen days after infusion, an analysis of the bone marrow of infused patients showed a significant increase in the following: (i) bone marrow cellularity, and (ii) myeloid, erythroid and megakaryocytic lineages at various stages of differentiation with a significant drop in fat cells and stromal cells; the predominant occupant of severe aplastic anaemia bone marrow

Bone marrow prior infusion showing reduced white and red blood cells cellularity

Bone marrow post infusion showing increased white and red blood cells cellularity

Significant increase in red blood cell mass (fig 3) in the bone marrow of patients infused with the retrodifferentiated stem cells with a corresponding increase in hemoglobin level as well as reticulocytes count.

Bone marrow prior infusion showing reduced red blood cell cellularity

Bone marrow post infusion showing increased red blood cell cellularity

• Significant steady increase in foetal heamoglobin as well as foetal red blood cells following infusion of the Retrodifferentiated stem cells. Foetal heamoglobin has a higher avidity to oxygen and is an important component in ameliorating sickle cell anaemia and some form of thalasemmia

• The retrodifferentiated pluripotent stem cells have exhibited a normal karyotype after infusion

• Transfusion-independence from infusion onwards

• The technology provided added side-benefits to aplastic/hypoplastic anaemia treatment: reduction of psoriasis lesions (fig 4) on the skin within 7 days and regeneration of bone tissue

Fig 4

Psoriasis lesion  prior infusion

Psoriasis lesion post infusion

⁽¹⁾Gluckman, E, Rocha, V, Boyer-Chammard, A, Locatelli, F et al. Outcome of cord blood transplantation from related and unrelated donors.  New Engl J Med.  1997: 6: 373-381