Poster/oral
Development of a bone marrow targeting peptide, using phage display, for stem cell
painting to treat after radioablative therapy
Thomas Kean1 (PhD), Lori Duesler1, Kazuki Sugahara2 (MD, PhD), Tambet Teesalu2 (PhD),
Erkki Ruoslahti2 (MD, PhD), Jim Dennis1 (PhD)
1) Case Western Reserve University, Dept Orthopaedics, Cleveland, Ohio
2) Burnham Institute for Medical Research at University of California, Santa Barbara, California
Problem – Radioablative therapy is used in several conditions including leukaemia. After
radiation treatment the patient is given a bone marrow transplant to restore their immune system
and hematopoietic potential. Engraftment of these cells within the bone marrow is inefficient and
could be enhanced by coating the cells with a targeting peptide. Therefore, targeting peptides
were sought using phage display.
Method – The right leg of mice was irradiated (250 rads) and the mouse allowed to recover for
24h. A T7 Phage library expressing a random 7 amino acid in its surface coat (1.28 x 109 peptide
sequences) was tail vein injected and allowed to circulate for 10 min. Organs (heart, liver, lung,
spleen and kidney) and bone marrow from each leg were then harvested. These were washed 4x
in PBS/BSA (1%), pellets were lysed, 0.9 ml of bacteria (BLT5403) added and incubated at RT
for 10 min. A dilution series was made, plaques counted and a phage titer calculated. Phage from
the right leg were amplified, purified then injected into the next mouse. This process was
repeated 5 times. Plaques from the legs and lung were picked, DNA amplified by PCR and
Table 1
Tissue
sequenced.
Phage ID
Results – A general increase in the number of phage isolated from
the bone marrow was seen in each successive round (Fig. 1). Phage
sequences and their frequency are displayed in Table 1. Selectivity
of the phage is demonstrated by the low crossover in each tissue.
Detailed analysis of the selected phage pool is underway.
yourname@uruni.edu
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
B11
B12
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
C17
L.Leg
1
1
2
1
4
4
1
1
1
1
1
1
1
R.Leg
lung
4
1
2
3
1
1
1
1
1
5
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1