U.S. patent application number 17/179212 was filed with the patent office on 2021-06-17 for enhanced reconstitution and autoreconstitution of the hematopoietic compartment.
The applicant listed for this patent is TAIGA BIOTECHNOLOGIES, INC.. Invention is credited to Gregory Alan BIRD, Yosef REFAELI, Brian C. TURNER.
Application Number | 20210177907 17/179212 |
Document ID | / |
Family ID | 1000005429649 |
Filed Date | 2021-06-17 |
United States Patent
Application |
20210177907 |
Kind Code |
A1 |
TURNER; Brian C. ; et
al. |
June 17, 2021 |
ENHANCED RECONSTITUTION AND AUTORECONSTITUTION OF THE HEMATOPOIETIC
COMPARTMENT
Abstract
The present disclosure relates to the acceleration of
hematopoietic compartment reconstitution in a subject in need of
hematopoietic stem cell transplantation by administering a
composition having a protein transduction domain-MYC (PTD-MYC)
fusion protein in combination with hematopoietic stem cell
transplantation and to the enhancement of hematopoietic compartment
autoreconstitution in a subject in need thereof by administering a
composition having a protein transduction domain-MYC (PTD-MYC)
fusion protein.
Inventors: |
TURNER; Brian C.; (Denver,
CO) ; REFAELI; Yosef; (Denver, CO) ; BIRD;
Gregory Alan; (Littleton, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TAIGA BIOTECHNOLOGIES, INC. |
Aurora |
CO |
US |
|
|
Family ID: |
1000005429649 |
Appl. No.: |
17/179212 |
Filed: |
February 18, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16261207 |
Jan 29, 2019 |
10953048 |
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17179212 |
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15785000 |
Oct 16, 2017 |
10206952 |
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16261207 |
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14415325 |
Jan 16, 2015 |
9789135 |
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PCT/US2013/051384 |
Jul 19, 2013 |
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15785000 |
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61785691 |
Mar 14, 2013 |
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61674224 |
Jul 20, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 35/28 20130101;
C07K 2319/10 20130101; C12N 5/0634 20130101; C07K 14/82 20130101;
C12N 5/0647 20130101; C07K 14/4747 20130101; C12N 2501/60 20130101;
A61K 38/1709 20130101; C07K 2319/21 20130101; A61K 38/1761
20130101; A61K 38/17 20130101; A61K 35/12 20130101; A61K 2035/124
20130101; C12N 2501/606 20130101; A61K 35/14 20130101; C07K 2319/40
20130101 |
International
Class: |
A61K 35/28 20060101
A61K035/28; A61K 38/17 20060101 A61K038/17; C07K 14/82 20060101
C07K014/82; C07K 14/47 20060101 C07K014/47; C12N 5/0789 20060101
C12N005/0789 |
Claims
1.-159. (canceled)
160. A method for preventing a decrease in hematopoietic
compartment cells in a subject in need thereof, the method
comprising administering a composition comprising a PTD-MYC fusion
protein to the subject.
161. The method of claim 160, wherein the hematopoietic compartment
cells comprise hematopoietic stem cells.
162. The method of claim 160, wherein the decrease in hematopoietic
compartment cells is due to chemotherapy or radiation therapy.
163. The method of claim 162, wherein the composition is to be
administered prior to chemotherapy or radiation therapy.
164. The method of claim 163, wherein the composition is to be
administered about 24 hours prior to the chemotherapy or radiation
therapy.
165. The method of claim 163, wherein the composition is to be
administered about 48 hours prior to the chemotherapy or radiation
therapy.
166. The method of claim 162, wherein the composition is to be
administered prior to and following the chemotherapy or radiation
therapy.
167. The method of claim 166, wherein the composition is to be
administered about 24 hours following chemotherapy or radiation
therapy.
168. The method of claim 161, wherein the decrease in hematopoietic
compartment cells is due to a bone marrow failure syndrome.
169. The method of claim 168, wherein the bone marrow failure
syndrome is aplastic anemia or Gulf War syndrome.
170. The method of claim 168, wherein the bone marrow failure
syndrome is an inherited bone marrow failure syndrome (IBMFS).
171. The method of claim 170, the IBMFS is selected from
amegakaryocytic thrombocytopenia, Diamond-Blackfan anemia,
dyskeratosis congenita, fanconi anemia, Pearson syndrome, severe
congenital neutropenia, Shwachman Diamond syndrome, and
thrombocytopenia absent radii, IVIC syndrome, WT syndrome,
radio-ulnar synostosis, and ataxia pancytopenia.
172. The method of claim 161, wherein the composition prevents a
decrease in the endogenous HSCs.
173. The method of claim 161, wherein the PTD-MYC fusion protein is
a TAT-MYC fusion protein.
174. The method of claim 161, wherein the PTD-MYC fusion protein is
to be administered at a dose of at least 0.1 mg/kg, at least 0.2
mg/kg, at least 0.3 mg/kg, at least 0.4 mg/kg, at least 0.5 mg/kg,
at least 0.6 mg/kg, at least 0.7 mg/kg, at least 0.8 mg/kg, at
least 0.9 mg/kg, at least 1 mg/kg, at least 2 mg/kg, at least 3
mg/kg, at least 4 mg/kg, at least 5 mg/kg, at least 6 mg/kg, at
least 7 mg/kg, at least 8 mg/kg, at least 9 mg/kg, at least 10
mg/kg, at least 20 mg/kg, at least 30 mg/kg, at least 40 mg/kg, or
at least 50 mg/kg of the subject's weight.
175. The method of claim 161, wherein the subject has cancer.
176. The method of claim 161, wherein the subject has a
hematological malignancy, a myeloma, multiple myeloma, a leukemia,
acute lymphoblastic leukemia, chronic lymphocytic leukemia, a
lymphoma, indolent lymphoma, non-Hodgkin lymphoma, diffuse B cell
lymphoma, follicular lymphoma, mantle cell lymphoma, T cell
lymphoma, Hodgkin lymphoma, a neuroblastoma, a retinoblastoma,
Shwachman Diamond syndrome, a brain tumor, Ewing's Sarcoma, a
Desmoplastic small round cell tumor, a relapsed germ cell tumor, a
hematological disorder, a hemoglobinopathy, an autoimmune disorder,
juvenile idiopathic arthritis, systemic lupus erythematosus, severe
combined immunodeficiency, congenital neutropenia with defective
stem cells, severe aplastic anemia, a sickle-cell disease, a
myelodysplasia syndrome, chronic granulomatous disease, a metabolic
disorder, Hurler syndrome, Gaucher disease, osteopetrosis,
malignant infantile osteopetrosis, heart disease, HIV, or AIDS.
177. The method of claim 161, wherein the subject is a human
subject.
178. A method of accelerating hematopoietic compartment
reconstitution after hematopoietic stem cell (HSC) transplantation
in a subject, the method comprising: a) administering a
therapeutically effective amount of a first composition comprising
HSCs to achieve hematopoietic compartment reconstitution in a
subject in need thereof; and b) administering a second composition
comprising a PTD-MYC fusion protein to the subject, wherein
administering the second composition achieves an acceleration in
hematopoietic compartment reconstitution compared to hematopoietic
compartment reconstitution in a subject that is not administered
the second composition.
179. The method of claim 178, wherein the a PTD-MYC fusion protein
is a TAT-MYC fusion protein.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/261,207, filed on Jan. 29, 2019, which is a
continuation of U.S. patent application Ser. No. 15/785,000, filed
on Oct. 16, 2017, now U.S. Pat. No. 10,206,952, which is a
continuation of U.S. patent application Ser. No. 14/415,325, filed
Jan. 16, 2015, now U.S. Pat. No. 9,789,135, which is a national
phase of International Application No. PCT/US2013/051384, filed
Jul. 19, 2013, which claims the benefit of U.S. Provisional
Application No. 61/674,224, filed Jul. 20, 2012, and U.S.
Provisional Application No. 61/785,691, filed Mar. 14, 2013, each
of which is hereby incorporated by reference in their entirety.
SUBMISSION OF SEQUENCE LISTING AS ASCII TEXT FILE
[0002] The instant application contains a Sequence Listing, which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Feb. 17, 2021, is named 106417-0540_Sequence_Listing.txt and is
10,740 bytes in size.
FIELD
[0003] The present disclosure generally relates to hematopoietic
compartment cell formation, cell survival, and cell proliferation
by hematopoietic stem cells. In particular, the present disclosure
relates to the acceleration of hematopoietic compartment
reconstitution and to the enhancement of hematopoietic compartment
autoreconstitution.
BACKGROUND
[0004] The use of hematopoietic stem cells for bone marrow
transplantation has revolutionized the approaches used to treat a
large number of hematological malignancies (e.g., leukemias), as
well as several widespread autoimmune diseases, and is a critical
treatment for immunodeficiencies (Buckley, RH, Annu Rev Immunol 22,
625-655, 2004). The use of hematopoietic stem cell transplantations
has also been successful in mitigating the effects of exposure to
high levels of radiation in several instances (Bishop, MR, Stem
Cells 15 Suppl 2, 305-310, 1997). In addition, hematopoietic stem
cell transplantations have been used to enable the administration
of high doses of cytotoxic chemotherapeutic agents to patients who
suffer from a number of solid organ tumors, thus enabling the
repopulation of the bone marrow following drug-induced toxicity
(Crivellari, G et al., Oncologist 12, 79-89, 2007). The use of
hematopoietic stem cell transplantation to improve the rate of
engraftment of solid organ transplantations is another recent
application of this medical procedure (Delis, S et al., Pancreas
32, 1-8, 2006). Recent studies also indicate that bone marrow
transplantation may have value in the treatment of heart disease
(Engelmann, M G et al., Curr Opin Mol Ther 8, 396-414, 2006).
Although the basis of this effect is unknown, these findings raise
the possibility that hematopoietic stem cells may be reprogrammed
to give rise to other tissues (Kiel, M J et al., Dev Biol 283,
29-39, 2005). Accordingly, adult hematopoietic stem cells may have
a much broader utility than, and may provide an alternative to,
controversial embryonic stem cell therapy. These therapeutic
applications of hematopoietic stem cell transplantation demonstrate
the medical and economic impact of improving hematopoietic stem
cell transplantation.
[0005] Several problems have limited the therapeutic application of
hematopoietic stem cell (HSC) transplantation. For example, one
major problem is the low number of HSCs available for transplants.
Patients who suffer from bone marrow failure, autoimmune diseases,
congenital immunodeficiencies, or hematological malignancies do not
provide a good source of HSCs for autologous transplantation
(Linker, C, Best Pract Res Clin Haematol 20, 77-84, 2007). In
addition, some bone marrow failure patients and cancer patients
require multiple rounds of HSC transplantation in order to achieve
full HSC engraftment following radiation or chemotherapy treatment
(Oliansky, D M et al., Biol Blood Marrow Transplant 13, 1-25,
2007). In cases where autologous HSC transplantation is not
possible, there is the additional problem of identifying an
appropriately histocompatible bone marrow donor. This is generally
accomplished using registries that have enrolled more than 6
million potential donors (de Mello, A N et al., J. Telemed.
Telecare 12 Suppl3, 64-6, 2006). Additionally, once selected, the
donor must undergo a grueling and painful process to mobilize HSCs
into the blood followed by 4-5 days of leukapheresis to isolate
rare long-term HSCs (Nervi, B et al., J Cell Biochem 99, 690-705,
2006). There have been a number of novel approaches aimed at
solving the problem of low numbers of HSCs available for
transplants, by expanding HSCs ex vivo after isolation, but the
ability to generate large numbers of long-term repopulating HSCs
that remain available and bioactive over a period of years has
remained elusive (Hoffman, R, Curr Opin Hematol 6, 184-91,
1999).
[0006] Another problem that limits the therapeutic application of
HSC transplantation is the time required for the transplanted HSCs
to reconstitute the functional and mature hematopoietic lineages
(i.e., the hematopoietic compartment) after the ablation of the
transplant recipient's resident immune system. One of the elements
required to promote the successful engraftment of transplanted HSCs
is the removal of the resident immune system. This is routinely
done by total body irradiation or chemical ablation. The end result
of this process is an almost completely immunocompromised patient
that is highly susceptible to opportunistic infection by
environmental microorganisms that humans routinely interact with
during normal activities, such as breathing and eating. This
problem is of greater concern with the significant increase in the
variety and heterogeneity of iatrogenic infectious agents, many of
which are highly resistant to existing antibiotics. The time
required for recovery of mature hematopoietic lineages after an HSC
transplant significantly affects the risk of the transplant
recipient developing and ultimately succumbing to opportunistic
infections.
[0007] The time required to repopulate mature hematopoietic
lineages in an HSC transplant recipient is affected by several
variables. One such variable is the differing recovery times
required to repopulate the different hematopoietic lineages
following an HSC transplant. For example, the myeloid compartment,
which is composed of monocytes, neutrophils, and basophils, usually
requires 4-8 weeks to recover following HSC transplantation. The
lymphoid compartment requires a significantly longer recovery time
in humans. For example, T-cells, NK cells, and NKT-cells require
between 4-8 months to recover, while B-cells require over 12 months
to recover in most individuals. The recovery time of the myeloid
lineage cells is critical for the minimal required defenses against
food-borne and environmental microorganisms. Another variable
affecting the time required to repopulate mature hematopoietic
lineages in an HSC transplant recipient is the number of HSCs
available for transplantation and the size/weight of the recipient.
A further variable is the nature of other treatments that an HSC
transplant patient may have been subjected to prior to the
transplantation. In most patients with some form of cancer, the
patient will have been given several rounds of chemotherapy prior
to receiving an HSC transplant. The use of such cytotoxic drugs can
impact the bone marrow niches to which the transplanted HSCs home
to and begin the differentiation process. In some instances where
the niches have been destroyed by cytotoxic drugs, several HSC
transplants may be required to initially reconstitute the niches
and subsequently seed the niches with pluripotent HSCs.
[0008] Moreover, the recovery time of mature hematopoietic lineages
following HSC transplantation is largely dependent of the ability
of the donor HSCs to find their way to bone marrow niches. Once the
HSCs arrive at the bone marrow niches, they need to establish a
molecular crosstalk with the niche-resident cells. This cross talk
is thought to regulate the nature and levels of cell-intrinsic
signals within the HSCs that regulate their survival,
proliferation, self-renewal, and differentiation. Thus, failure of
HSCs to find their way to bone marrow niches, home properly, or
correctly establish such molecular crosstalk with the niches can
result in HSC engraftment failure. Additionally, failure of HSCs to
find their way to bone marrow niches can also result in only a
short term recovery of mature hematopoietic lineages or only a
partial reconstitution of mature hematopoietic lineages that will
slowly subside as a result of long-term bone marrow failure.
[0009] The lag time between ablation of a patient's resident immune
system and hematopoietic lineage reconstitution by HSC
transplantation is thus one of the major risk factors for the
development of potentially fatal complications, such as
opportunistic infections or HSC engraftment failure. Several
approaches have been attempted to decrease the time required to
reconstitute mature hematopoietic lineages following HSC
transplantation. Examples of such approaches include the use of
higher numbers of HSCs for transplantation, partial resident immune
system ablation and multiple transplants of smaller numbers of
HSCs, pre-conditioning of donor bone marrow to retain its resident
T-cells, growth factor treatment of the patient following HSC
transplantation, and the use of monoclonal antibodies and/or small
molecule modifiers that target enzymes affecting E-selectin
expression in order to improve HSC homing to bone marrow niches
after HSC transplantation (Adams GB and Scadden DT, Gene Ther 15,
96-99, 2008; Campbell TB and Broxmeyer HE, Front. Biosc. 13,
1795-1805, 2008; Rocha V and Boxmeyer H, Biology of Bone marrow
transplantation 16, S126-S132, 2009; and Hogatt J. et al., Blood
113, 5444-55, 2009). However, these solutions only provide a modest
improvement over current approaches without significantly affecting
the frequency of fatal opportunistic infections in the patient
population.
BRIEF SUMMARY
[0010] Accordingly, there is a need for improved approaches for
accelerating hematopoietic compartment reconstitution in a
hematopoietic stem cell (HSC) transplant recipient that result in
one or more of the following: increase the number of HSCs available
for transplantation, increase the number of HSCs that productively
home to bone marrow niches, reduce the risk of opportunistic
infections, and reduce the risk of HSC engraftment failure. There
is also a need for improved approaches for enhancing hematopoietic
compartment autoreconstitution in a subject in need of
hematopoietic compartment autoreconstitution.
[0011] In order to meet the above needs, the present disclosure
provides novel methods of enhancing hematopoietic compartment
reconstitution (e.g., HSC engraftment) in a subject by treating a
population of HSCs with a MYC-composition, such as a protein
transduction domain-MYC (PTD-MYC) fusion protein, a
Bcl-2-composition, such as a PTD-Bcl-2 fusion protein, or both,
prior to transplanting the HSCs into the subject; and to novel
methods of enhancing hematopoietic compartment cell formation in a
subject by administering a composition having a MYC-composition,
such as a protein transduction domain-MYC (PTD-MYC) fusion protein,
a Bcl-2-composition, such as a protein transduction domain-Bcl-2
(PTD-Bcl-2) fusion protein, or both. Certain aspects relate to
methods of accelerating hematopoietic compartment reconstitution in
a hematopoietic stem cell (HSC) transplant recipient by
administering a composition having a MYC-composition, such as a
protein transduction domain-MYC (PTD-MYC) fusion protein, a
Bcl-2-composition, such as a protein transduction domain-Bcl-2
(PTD-Bcl-2) fusion protein, or both in combination with the HSC
transplantation. Advantageously, such novel methods of accelerating
hematopoietic compartment reconstitution may increase the number of
HSCs that productively home to bone marrow niches of the transplant
recipient and/or may enhance the rate of myeloid and lymphoid
compartment reconstitution, thereby reducing the risk of
opportunistic infections in the transplant recipient and reducing
the risk of HSC engraftment failure.
[0012] Additionally, the present disclosure is based, at least in
part, on the surprising discovery that administering a composition
having a fusion protein containing a MYC polypeptide and a protein
transduction domain (PTD), such as the HIV TAT protein transduction
domain, (TAT-MYC fusion protein) after HSC transplantation
accelerates the recovery time of mature hematopoietic lineages in
an HSC transplant recipient. Without wishing to be bound by theory,
it is believed that administering a MYC-composition, such as a
PTD-MYC fusion protein to an HSC transplant recipient increases MYC
activity in the hematopoietic compartment of the recipient,
resulting in enhanced HSC homing to bone marrow niches in the
recipient, and enhanced survival of the HSCs after transplantation
resulting in a higher likelihood of HSC homing. Enhanced HSC homing
improves the number of transplanted HSCs that can successfully
interact with the cell matrix at bone marrow niches (i.e.,
productive homing), improves the number of transplanted HSCs at
bone marrow niches that self-renew and produce the relevant
progenitor cell types, and improves the kinetics of homing to bone
marrow niches, which results in the acceleration of hematopoietic
compartment reconstitution. Without wishing to be bound by theory,
it is also believed that administering a MYC-composition, such as a
PTD-MYC fusion protein to an HSC transplant recipient increases MYC
activity in the transplanted HSCs, which increases the number of
HSCs that self-renew and produce the relevant progenitor cell types
and subtypes for reconstituting the functional and mature
hematopoietic lineages (i.e., the hematopoietic compartment).
Moreover, treating HSCs to be transplanted with a MYC-composition,
such as a PTD-MYC fusion protein approximately from about 10
minutes to about 24 hours prior to transplantation also accelerates
the recovery time of mature hematopoietic lineages in an HSC
transplant recipient. In some embodiments, the amount of time
required to transduce or otherwise introduce the MYC-composition
into the HSCs is sufficient to achieve an acceleration in the
recovery time of mature hematopoietic lineages in an HSC transplant
recipient.
[0013] Other aspects of the present disclosure also relate to novel
methods of enhancing hematopoietic compartment autoreconstitution
in a subject in need of hematopoietic compartment
autoreconstitution by administering a composition having a
MYC-composition, such as a protein transduction domain-MYC
(PTD-MYC) fusion protein, a Bcl-2-composition, such as a protein
transduction domain-Bcl-2 (PTD-Bcl-2) fusion protein, or both,
which induce endogenous HSCs to autoreconstitute the hematopoietic
compartment in the subject. Advantageously, such novel methods of
enhancing hematopoietic compartment autoreconstitution may protect
patients, or facilitate recovery, from insults to the hematopoietic
compartment, such as chemotherapy and radiation therapy. Such novel
methods may also be used to treat bone marrow failure syndromes.
Without wishing to be bound by theory, it is believed that
administering a MYC-composition, such as a PTD-MYC fusion protein,
a Bcl-2-composition, such as a PTD-Bcl-2 fusion protein, or both to
a subject in need of hematopoietic compartment autoreconstitution
increases MYC activity, Bcl-2 activity, or both in the endogenous
HSCs, which increases the number of HSCs that self-renew and
produce the relevant progenitor cell types and subtypes for
autoreconstituting the functional and mature hematopoietic lineages
(i.e., the hematopoietic compartment). Furthermore, without wishing
to be bound by theory, it is also believed that MYC activity also
enhances HSC localization to bone marrow niches.
[0014] While Refaeli et al. state that the protooncogene MYC can
break B cell tolerance (Refaeli et al. Proc Nat Acad Sci 102(11):
4097-4102, 2005), Refaeli et al. provide no guidance regarding the
use of PTD-MYC fusion proteins, or the role of MYC in enhancing the
ability of HSCs to form hematopoietic compartment cells. Moreover,
while US2007/0116691, US2010/0297763, WO2007/047583,
US2010/0047217, and WO2010/011644 disclose conditionally
immortalized long-term stem cells and methods for preparing
differentiated cells, none of these applications disclose that MYC
enhances the ability of exogenously added HSCs to reconstitute the
hematopoietic compartment in an HSC transplant recipient, or the
ability of endogenous HSCs to autoreconstitute the hematopoietic
compartment in a subject.
[0015] In contrast to Refaeli et al., US2007/0116691,
US2010/0297763, WO2007/047583, US2010/0047217, and WO2010/011644,
the inventors have surprisingly shown that administering a
MYC-composition, such as a PTD-MYC fusion protein in HSC transplant
recipients 24 hours after accelerates hematopoietic compartment
reconstitution by reducing the time required for recovery of mature
hematopoietic lineages by at least 50%. The inventors have also
surprisingly shown that administering a MYC-composition, such as a
PTD-MYC fusion protein in a subject having a reduction in their
hematopoietic compartment enhances autoreconstitution in the
subject.
[0016] Accordingly, the present disclosure relates to a method of
enhancing hematopoietic compartment reconstitution to a subject in
need of hematopoietic stem cell transplantation, by: treating a
population of hematopoietic stem cells with a composition
containing a MYC-composition, a composition containing a
Bcl-2-composition, or both, for less than about 13 days; and
administering to the subject, a therapeutically effective amount of
the treated population of hematopoietic stem cells to reconstitute
the hematopoietic compartment of the subject, wherein hematopoietic
compartment reconstitution is enhanced compared to hematopoietic
compartment reconstitution in a subject that is administered a
population of hematopoietic stem cells that were not treated with
the composition containing a MYC-composition, the composition
containing a Bcl-2-composition, or both. Advantageously, a
composition of the present disclosure containing a MYC-composition,
a Bcl-2 composition, or both may also be administered to the
subject receiving the pre-treated hematopoietic stem cells to
maintain and/or further enhance hematopoietic compartment
reconstitution in the subject.
[0017] In certain embodiments that may be combined with any of the
preceding embodiments, the population of hematopoietic stem cells
is treated with the composition containing a MYC-composition, the
composition containing a Bcl-2-composition, or both, for less than
about 12 days, less than about 11 days, less than about 10 days,
less than about 9 days, less than about 8 days, less than about 7
days, less than about 6 days, less than about 5 days, less than
about 4 days, less than about 2 days, or less than about 1 day. In
certain embodiments that may be combined with any of the preceding
embodiments, the population of hematopoietic stem cells is treated
with the composition containing a MYC-composition, the composition
containing a Bcl-2-composition, or both, for less than about 24
hours, less than about 23 hours, less than about 22 hours, less
than about 21 hours, less than about 20 hours, less than about 19
hours, less than about 18 hours, less than about 17 hours, less
than about 16 hours, less than about 15 hours, less than about 14
hours, less than about 13 hours, less than about 12 hours, less
than about 11 hours, less than about 10 hours, less than about 9
hours, less than about 8 hours, less than about 7 hours, less than
about 6 hours, less than about 5 hours, less than about 4 hours,
less than about 3 hours, less than about 2 hours, or less than
about 1 hour. In certain embodiments that may be combined with any
of the preceding embodiments, the population of hematopoietic stem
cells is treated with the composition containing a MYC-composition,
the composition containing a Bcl-2-composition, or both, for less
than about 60 minutes, less than about 55 minutes, less than about
50 minutes, less than about 45 minutes, less than about 40 minutes,
less than about 35 minutes, less than about 30 minutes, less than
about 29 minutes, less than about 28 minutes, less than about 27
minutes, less than about 26 minutes, less than about 25 minutes,
less than about 24 minutes, less than about 23 minutes, less than
about 22 minutes, less than about 21 minutes, less than about 20
minutes, less than about 19 minutes, less than about 18 minutes,
less than about 17 minutes, less than about 16 minutes, less than
about 15 minutes, less than about 14 minutes, less than about 13
minutes, less than about 12 minutes, less than about 11 minutes, or
less than about 10 minutes. In certain embodiments that may be
combined with any of the preceding embodiments, the population of
hematopoietic stem cells is treated with the composition containing
a MYC-composition. In certain embodiments that may be combined with
any of the preceding embodiments, the population of hematopoietic
stem cells is treated with the composition containing a
Bel-2-composition. In certain embodiments that may be combined with
any of the preceding embodiments, the population of hematopoietic
stem cells is treated with the composition containing a
MYC-composition and the composition containing a Bcl-2-composition.
In certain embodiments that may be combined with any of the
preceding embodiments, the therapeutically effective amount of the
composition containing a MYC-composition is at least 0.5.mu./ml, at
least 0.6.mu./ml, at least 0.7.mu./ml, at least 0.8.mu./ml, at
least 0.9.mu./ml, at least 1.mu./ml, at least 2.mu./ml, at least
3.mu./ml, at least 4.mu./ml, at least 5.mu./ml, at least 6.mu./ml,
at least 7.mu./ml, at least 8.mu./ml, at least 9.mu./ml, at least
10.mu./ml, at least 15.mu./ml, at least 20.mu./ml, at least
25.mu./ml, at least 30.mu./ml, at least 35.mu./ml, at least
40.mu./ml, at least 45.mu./ml, at least 50.mu./ml, at least
55.mu./ml, at least 60.mu./ml, at least 65.mu./ml, at least
70.mu./ml, at least 75.mu./ml, at least 80.mu./ml, at least
85.mu./ml, at least 90.mu./ml, at least 95.mu./ml, or at least
100.mu./ml. In certain embodiments that may be combined with any of
the preceding embodiments, the Bcl-2-composition contains a Bcl-2
polypeptide, a homologue thereof, an analogue thereof, or a
biologically active fragment thereof. In certain embodiments that
may be combined with any of the preceding embodiments, the
Bcl-2-composition contains a protein transduction domain (PTD). In
certain embodiments that may be combined with any of the preceding
embodiments, the Bcl-2-composition is a PTD-Bcl-2 fusion protein.
In certain embodiments that may be combined with any of the
preceding embodiments, the Bcl-2-composition is a TAT-Bcl-2 fusion
protein. In certain embodiments that may be combined with any of
the preceding embodiments, the therapeutically effective amount of
the composition containing a Bcl-2-composition is at least
0.5.mu./ml, at least 0.6.mu./ml, at least 0.7.mu./ml, at least
0.8.mu./ml, at least 0.9.mu./ml, at least 1.mu./ml, at least
2.mu./ml, at least 3.mu./ml, at least 4.mu./ml, at least 5.mu./ml,
at least 6.mu./ml, at least 7.mu./ml, at least 8.mu./ml, at least
9.mu./ml, at least 10.mu./ml, at least 15.mu./ml, at least
20.mu./ml, at least 25.mu./ml, at least 30.mu./ml, at least
35.mu./ml, at least 40.mu./ml, at least 45.mu./ml, at least
50.mu./ml, at least 55.mu./ml, at least 60.mu./ml, at least
65.mu./ml, at least 70.mu./ml, at least 75.mu./ml, at least
80.mu./ml, at least 85.mu./ml, at least 90.mu./ml, at least
95.mu./ml, or at least 100.mu./ml. In certain embodiments that may
be combined with any of the preceding embodiments, the composition
containing a MYC-composition further contains a pharmaceutically
acceptable carrier. In certain embodiments that may be combined
with any of the preceding embodiments, the population of
hematopoietic stem cells is washed prior to being administered to
the subject in need thereof. In certain embodiments that may be
combined with any of the preceding embodiments, the population of
hematopoietic stem cells is administered to the subject in need
thereof without washing the population of hematopoietic stem cells.
In certain embodiments that may be combined with any of the
preceding embodiments, the subject has or had a hematological
malignancy, a myeloma, multiple myeloma, a leukemia, acute
lymphoblastic leukemia, chronic lymphocytic leukemia, a lymphoma,
indolent lymphoma, non-Hodgkin lymphoma, diffuse B cell lymphoma,
follicular lymphoma, mantle cell lymphoma, T cell lymphoma, Hodgkin
lymphoma, a neuroblastoma, a retinoblastoma, Shwachman Diamond
syndrome, a brain tumor, Ewing's Sarcoma, a Desmoplastic small
round cell tumor, a relapsed germ cell tumor, a hematological
disorder, a hemoglobinopathy, an autoimmune disorder, juvenile
idiopathic arthritis, systemic lupus erythematosus, severe combined
immunodeficiency, congenital neutropenia with defective stem cells,
severe aplastic anemia, a sickle-cell disease, a myelodysplastic
syndrome, chronic granulomatous disease, a metabolic disorder,
Hurler syndrome, Gaucher disease, osteopetrosis, malignant
infantile osteopetrosis, heart disease, HIV, or AIDS. In certain
embodiments that may be combined with any of the preceding
embodiments, the subject has had an organ transplant. In certain
embodiments that may be combined with any of the preceding
embodiments, the population of hematopoietic stem cells were
obtained from bone marrow, from peripheral blood cells, from
peripheral blood cells that have undergone apheresis, from
peripheral blood cells that have undergone leukapheresis, from
umbilical cord blood, from amniotic fluid, from cultured HSC cells,
from an immortalized HSC cell line, or from a conditionally
immortalized HSC cell line. In certain embodiments that may be
combined with any of the preceding embodiments, the treated
population of hematopoietic stem cells is administered as a step in
a hematopoietic stem cell (HSC) transplantation procedure. In
certain embodiments that may be combined with any of the preceding
embodiments, the HSC transplantation procedure is a myeloablative
HSC transplantation procedure. In certain embodiments that may be
combined with any of the preceding embodiments, the HSC
transplantation procedure is a non-myeloablative HSC
transplantation procedure. In certain embodiments that may be
combined with any of the preceding embodiments, the HSC
transplantation is an autologous HSC transplantation or an
allogenic HSC transplantation. In certain embodiments that may be
combined with any of the preceding embodiments, administration of
the treated population of hematopoietic stem cells accelerates
hematopoietic compartment reconstitution after HSC transplantation
in the subject. In certain embodiments that may be combined with
any of the preceding embodiments, administering the treated
population of hematopoietic stem cells achieves an at least 50%
acceleration in hematopoietic compartment reconstitution, compared
to hematopoietic compartment reconstitution in a subject that is
administered a population of hematopoietic stem cells that were not
treated with the composition containing a MYC-composition, the
composition containing a Bcl-2-composition, or both. In certain
embodiments that may be combined with any of the preceding
embodiments, the accelerated hematopoietic compartment
reconstitution in the subject results in T cell compartment
reconstitution that is accelerated by at least 50%, compared to T
cell reconstitution in a subject that is administered a population
of hematopoietic stem cells that were not treated with the
composition containing a MYC-composition, the composition
containing a Bcl-2-composition, or both. In certain embodiments
that may be combined with any of the preceding embodiments, the
accelerated hematopoietic compartment reconstitution in the subject
results in B cell compartment reconstitution that is accelerated by
at least 50%, compared to B cell reconstitution in a subject that
is administered a population of hematopoietic stem cells that were
not treated with the composition containing a MYC-composition, the
composition containing a Bcl-2-composition, or both. In certain
embodiments that may be combined with any of the preceding
embodiments, the accelerated hematopoietic compartment
reconstitution in the subject results in NK-cell compartment
reconstitution that is accelerated by at least 50%, compared to
NK-cell reconstitution in a subject that is administered a
population of hematopoietic stem cells that were not treated with
the composition containing a MYC-composition, the composition
containing a Bcl-2-composition, or both. In certain embodiments
that may be combined with any of the preceding embodiments, he
accelerated hematopoietic compartment reconstitution in the subject
results in myeloid cell compartment reconstitution that is
accelerated by at least 50%, compared to myeloid cell compartment
reconstitution in a subject that is administered a population of
hematopoietic stem cells that were not treated with the composition
containing a MYC-composition, the composition containing a
Bcl-2-composition, or both. In certain embodiments that may be
combined with any of the preceding embodiments, the accelerated
hematopoietic compartment reconstitution in the subject results in
neutrophil recovery that is accelerated by at least 50%, compared
to neutrophil recovery in a subject that is administered a
population of hematopoietic stem cells that were not treated with
the composition containing a MYC-composition, the composition
containing a Bcl-2-composition, or both. In certain embodiments
that may be combined with any of the preceding embodiments,
administration of the composition containing a MYC-composition
results in a 50% increase in HSC productive homing to bone marrow
niches in the subject, compared to a subject that is administered a
population of hematopoietic stem cells that were not treated with
the composition containing a MYC-composition, the composition
containing a Bcl-2-composition, or both. In certain embodiments
that may be combined with any of the preceding embodiments, further
including administering a third composition containing a
MYC-composition, a Bcl-2-composition, or both; and optionally at
least one cytokine, growth factor, antibody, and/or small molecule
modifier. In certain embodiments that may be combined with any of
the preceding embodiments, the third composition further includes a
pharmaceutically acceptable carrier. In certain embodiments that
may be combined with any of the preceding embodiments, the
population of hematopoietic stem cells is a population of human
hematopoietic stem cells. In certain embodiments that may be
combined with any of the preceding embodiments, the MYC-composition
contains a MYC polypeptide, a homologue thereof, an analogue
thereof, or a biologically active fragment thereof. In certain
embodiments that may be combined with any of the preceding
embodiments, the MYC-compound contains a protein transduction
domain (PTD). In certain embodiments that may be combined with any
of the preceding embodiments, the MYC-composition is a PTD-MYC
fusion protein. In certain embodiments that may be combined with
any of the preceding embodiments, the MYC-composition is a TAT-MYC
fusion protein. In certain embodiments that may be combined with
any of the preceding embodiments, the subject is a human patient.
In certain embodiments that may be combined with any of the
preceding embodiments, the subject is a non-human animal.
[0018] Other aspects of the present disclosure relate to a method
of enhancing hematopoietic compartment cell formation in a subject,
by: administering a therapeutically effective amount of a
composition containing a MYC-composition, a Bcl-2-composition, or
both to a subject in need thereof, where hematopoietic compartment
formation is enhanced compared to hematopoietic compartment
formation in a subject that is not administered the
composition.
In certain embodiments that may be combined with any of the
preceding embodiments, the composition contains a MYC-composition.
In certain embodiments that may be combined with any of the
preceding embodiments, the composition contains a
Bcl-2-composition. In certain embodiments that may be combined with
any of the preceding embodiments, the composition contains a
MYC-composition and a Bcl-2-composition. In certain embodiments
that may be combined with any of the preceding embodiments, the
Bcl-2-composition contains a Bcl-2 polypeptide, a homologue
thereof, an analogue thereof, or a biologically active fragment
thereof. In certain embodiments that may be combined with any of
the preceding embodiments, the Bcl-2-composition comprises a
protein transduction domain (PTD). In certain embodiments that may
be combined with any of the preceding embodiments, the
Bcl-2-composition is a PTD-Bcl-2 fusion protein. In certain
embodiments that may be combined with any of the preceding
embodiments, the Bcl-2-composition is a TAT-Bcl-2 fusion protein.
In certain embodiments that may be combined with any of the
preceding embodiments, the MYC-composition contains a MYC
polypeptide, a homologue thereof, an analogue thereof, or a
biologically active fragment thereof. In certain embodiments that
may be combined with any of the preceding embodiments, the
MYC-compound contains a protein transduction domain (PTD). In
certain embodiments that may be combined with any of the preceding
embodiments, the MYC-composition is a PTD-MYC fusion protein. In
certain embodiments that may be combined with any of the preceding
embodiments, the MYC-composition is a TAT-MYC fusion protein. In
certain embodiments that may be combined with any of the preceding
embodiments, the subject is in need or was in need of hematopoietic
stem cell (HSC) transplantation. In certain embodiments, the
subject has or had a hematological malignancy, a myeloma, multiple
myeloma, a leukemia, acute lymphoblastic leukemia, chronic
lymphocytic leukemia, a lymphoma, indolent lymphoma, non-Hodgkin
lymphoma, diffuse B cell lymphoma, follicular lymphoma, mantle cell
lymphoma, T cell lymphoma, Hodgkin lymphoma, a neuroblastoma, a
retinoblastoma, Shwachman Diamond syndrome, a brain tumor, Ewing's
Sarcoma, a Desmoplastic small round cell tumor, a relapsed germ
cell tumor, a hematological disorder, a hemoglobinopathy, an
autoimmune disorder, juvenile idiopathic arthritis, systemic lupus
erythematosus, severe combined immunodeficiency, congenital
neutropenia with defective stem cells, severe aplastic anemia, a
sickle-cell disease, a myelodysplastic syndrome, chronic
granulomatous disease, a metabolic disorder, Hurler syndrome,
Gaucher disease, osteopetrosis, malignant infantile osteopetrosis,
heart disease, HIV, or AIDS. In certain embodiments, the subject
has had an organ transplant. In certain embodiments that may be
combined with any of the preceding embodiments, the method further
includes administering a therapeutically effective amount of a
second composition containing HSCs to achieve hematopoietic
compartment reconstitution in the subject. In certain embodiments,
the composition containing a MYC-composition, a Bcl-2-composition,
or both, a Bcl-2-composition, or both is administered before,
after, or concurrently with the administering of the second
composition. In certain embodiments, the composition containing a
MYC-composition, a Bcl-2-composition, or both, a Bcl-2-composition,
or both is administered at least 5 days, at least 4 days, at least
3 days, at least 2 days, or at least 1 day before the administering
of the second composition. In certain embodiments, the composition
containing a MYC-composition, a Bcl-2-composition, or both, a
Bcl-2-composition, or both is administered concurrently with the
administering of the second composition. In certain embodiments,
the composition containing a MYC-composition, a Bcl-2-composition,
or both, a Bcl-2-composition, or both is administered at least 1
day, at least 2 days, at least three days, at least 4 days, at
least 5 days, at least 6 days, at least 1 week, at least 2 weeks,
or at least 3 weeks after the administering of the second
composition. In certain embodiments that may be combined with any
of the preceding embodiments, administration of the composition
containing a MYC-composition, a Bcl-2-composition, or both, a
Bcl-2-composition, or both results in an expansion of the HSCs
contained in the second composition. In certain embodiments that
may be combined with any of the preceding embodiments, the second
composition containing HSCs was cultured in the presence of a
MYC-composition, a Bcl-2-composition, or both, before the
administering of the second composition. In certain embodiments,
culturing the second composition in the presence of the
MYC-composition, Bcl-2-composition, or both conditionally
immortalized the HSCs. In certain embodiments, immortalization of
the HSCs resulted in an expansion of the HSCs. In certain
embodiments that may be combined with any of the preceding
embodiments, the second composition as administered includes the
MYC-composition, Bcl-2-composition, or both as a result of being
cultured in the presence of the MYC-composition, Bcl-2-composition,
or both. In certain embodiments that may be combined with any of
the preceding embodiments, the HSCs were obtained from bone marrow,
from peripheral blood cells, from peripheral blood cells that have
undergone apheresis, from peripheral blood cells that have
undergone leukapheresis, from umbilical cord blood, from amniotic
fluid, from cultured HSC cells, from an immortalized HSC cell line,
or from a conditionally immortalized HSC cell line. In certain
embodiments that may be combined with any of the preceding
embodiments, the HSCs are present in bone marrow, in peripheral
blood cells, in peripheral blood cells that have undergone
apheresis, in peripheral blood cells that have undergone
leukapheresis, in umbilical cord blood, or in amniotic fluid. In
certain embodiments that may be combined with any of the preceding
embodiments, the second composition is administered as a step in an
HSC transplantation procedure. In certain embodiments, the HSC
transplantation procedure is a myeloablative HSC transplantation
procedure. In certain embodiments, the HSC transplantation
procedure is a non-myeloablative HSC transplantation procedure. In
certain embodiments that may be combined with any of the preceding
embodiments, the HSC transplantation is an autologous HSC
transplantation or an allogenic HSC transplantation. In certain
embodiments that may be combined with any of the preceding
embodiments, administration of the composition containing a
MYC-composition, a Bcl-2-composition, or both, a Bcl-2-composition,
or both accelerates hematopoietic compartment reconstitution after
HSC transplantation in the subject. In certain embodiments,
administering the composition containing a MYC-composition, a
Bcl-2-composition, or both, a Bcl-2-composition, or both achieves
an at least 50% acceleration in hematopoietic compartment
reconstitution compared to hematopoietic compartment reconstitution
in a subject that is not administered the composition. In certain
embodiments that may be combined with any of the preceding
embodiments, the accelerated hematopoietic compartment
reconstitution in the subject results in T cell compartment
reconstitution that is accelerated by at least 50% compared to T
cell reconstitution in a subject that is not administered the
composition. In certain embodiments that may be combined with any
of the preceding embodiments, the accelerated hematopoietic
compartment reconstitution in the subject results in B cell
compartment reconstitution that is accelerated by at least 50%
compared to B cell reconstitution in a subject that is not
administered the composition. In certain embodiments that may be
combined with any of the preceding embodiments, the accelerated
hematopoietic compartment reconstitution in the subject results in
NK-cell compartment reconstitution that is accelerated by at least
50% compared to NK-cell reconstitution in a subject that is not
administered the composition. In certain embodiments that may be
combined with any of the preceding embodiments, the accelerated
hematopoietic compartment reconstitution in the subject results in
myeloid cell compartment reconstitution that is accelerated by at
least 50% compared to myeloid cell compartment reconstitution in a
subject that is not administered the composition. In certain
embodiments that may be combined with any of the preceding
embodiments, the accelerated hematopoietic compartment
reconstitution in the subject results in neutrophil recovery that
is accelerated by at least 50% compared to neutrophil recovery in a
subject that is not administered the composition. In certain
embodiments that may be combined with any of the preceding
embodiments, administration of the composition containing a
MYC-composition, a Bcl-2-composition, or both, a Bcl-2-composition,
or both results in a 50% increase in HSC productive homing to bone
marrow niches in the subject. In certain embodiments that may be
combined with any of the preceding embodiments, the therapeutically
effective amount of the second composition administered to the
subject is less when the composition containing a MYC-composition,
a Bcl-2-composition, or both, a Bcl-2-composition, or both is
administered compared to the amount required when the composition
containing a MYC-composition, a Bcl-2-composition, or both, a
Bcl-2-composition, or both is not administered. In certain
embodiments that may be combined with any of the preceding
embodiments, further including administering a third composition
containing at least one cytokine, growth factor, antibody, and/or
small molecule modifier. In certain embodiments, the third
composition further contains a pharmaceutically acceptable carrier.
In certain embodiments that may be combined with any of the
preceding embodiments, the HSCs contained in the second composition
are human HSCs. In certain embodiments, the subject is in need or
was in need of hematopoietic compartment autoreconstitution. In
certain embodiments, the MYC-composition, Bcl-2-composition, or
both induces endogenous hematopoietic stem cells (HSCs) to
autoreconstitute the hematopoietic compartment in the subject. In
certain embodiments that may be combined with any of the preceding
embodiments, the hematopoietic compartment autoreconstitution is
enhanced compared to hematopoietic compartment autoreconstitution
in a subject that is not administered the composition containing a
MYC-composition, a Bcl-2-composition, or both, a Bcl-2-composition,
or both. In certain embodiments that may be combined with any of
the preceding embodiments, the enhanced hematopoietic compartment
autoreconstitution in the subject results in enhanced T cell
compartment autoreconstitution, enhanced B cell compartment
autoreconstitution, enhanced NK-cell compartment
autoreconstitution, enhanced myeloid cell compartment
autoreconstitution, or neutrophil recovery. In certain embodiments
that may be combined with any of the preceding embodiments,
administration of the composition containing a MYC-composition, a
Bcl-2-composition, or both, a Bcl-2-composition, or both results in
an expansion of endogenous HSCs. In certain embodiments that may be
combined with any of the preceding embodiments, the subject is
undergoing or has undergone chemotherapy. In certain embodiments,
administration of the composition containing a MYC-composition, a
Bcl-2-composition, or both, a Bcl-2-composition, or both prevents a
decrease in hematopoietic compartment cells due to the
chemotherapy. In certain embodiments that may be combined with any
of the preceding embodiments, administration of the composition
containing a MYC-composition, a Bcl-2-composition, or both prevents
a decrease in the amount of endogenous HSCs due to the
chemotherapy. In certain embodiments that may be combined with any
of the preceding embodiments, the subject is undergoing or has
undergone radiation therapy. In certain embodiments, administration
of the composition containing a MYC-composition, a
Bcl-2-composition, or both prevents a decrease in hematopoietic
compartment cells due to the radiation therapy. In certain
embodiments that may be combined with any of the preceding
embodiments, administration of the composition containing a
MYC-composition, a Bcl-2-composition, or both prevents a decrease
in the amount of endogenous HSCs due to the radiation therapy. In
certain embodiments that may be combined with any of the preceding
embodiments, the subject has a bone marrow failure syndrome. In
certain embodiments, the bone marrow failure syndrome is aplastic
anemia or Gulf War syndrome. In certain embodiments, the bone
marrow failure syndrome is an inherited bone marrow failure
syndrome (IBMFS). In certain embodiments, the IBMFS is selected
from amegakaryocytic thrombocytopenia, Diamond-Blackfan anemia,
dyskeratosis congenita, fanconi anemia, Pearson syndrome, severe
congenital neutropenia, Shwachman-Diamond syndrome, and
thrombocytopenia absent radii, IVIC syndrome, WT syndrome,
radio-ulnar synostosis, and ataxia pancytopenia. In certain
embodiments that may be combined with any of the preceding
embodiments, administration of the composition containing a
MYC-composition, a Bcl-2-composition, or both prevents a decrease
in hematopoietic compartment cells due to the bone marrow failure
syndrome. In certain embodiments that may be combined with any of
the preceding embodiments, administration of the composition
containing a MYC-composition, a Bcl-2-composition, or both prevents
a decrease in the amount of endogenous HSCs due to the bone marrow
failure syndrome. In certain embodiments that may be combined with
any of the preceding embodiments, the therapeutically effective
amount of the composition containing a MYC-composition is at least
0.1 mg/kg, at least 0.2 mg/kg, at least 0.3 mg/kg, at least 0.4
mg/kg, at least 0.5 mg/kg, at least 0.6 mg/kg, at least 0.7 mg/kg,
at least 0.8 mg/kg, at least 0.9 mg/kg, at least 1 mg/kg, at least
2 mg/kg, at least 3 mg/kg, at least 4 mg/kg, at least 5 mg/kg, at
least 6 mg/kg, at least 7 mg/kg, at least 8 mg/kg, at least 9
mg/kg, at least 10 mg/kg, at least 20 mg/kg, at least 30 mg/kg, at
least 40 mg/kg, or at least 50 mg/kg of the subject's weight. In
certain embodiments that may be combined with any of the preceding
embodiments, the therapeutically effective amount of the
composition comprising a Bcl-2-composition is at least 0.1 mg/kg,
at least 0.2 mg/kg, at least 0.3 mg/kg, at least 0.4 mg/kg, at
least 0.5 mg/kg, at least 0.6 mg/kg, at least 0.7 mg/kg, at least
0.8 mg/kg, at least 0.9 mg/kg, at least 1 mg/kg, at least 2 mg/kg,
at least 3 mg/kg, at least 4 mg/kg, at least 5 mg/kg, at least 6
mg/kg, at least 7 mg/kg, at least 8 mg/kg, at least 9 mg/kg, at
least 10 mg/kg, at least 20 mg/kg, at least 30 mg/kg, at least 40
mg/kg, or at least 50 mg/kg of the subject's weight. In certain
embodiments that may be combined with any of the preceding
embodiments, the composition containing a MYC-composition, a
Bcl-2-composition, or both further contains a pharmaceutically
acceptable carrier. In certain embodiments that may be combined
with any of the preceding embodiments, the subject is a human
patient. In certain embodiments that may be combined with any of
the preceding embodiments, the subject is a non-human animal.
[0020] Other aspects of the present disclosure relate to a method
of accelerating hematopoietic compartment reconstitution after
hematopoietic stem cell (HSC) transplantation in a subject, by: a)
administering a therapeutically effective amount of a first
composition containing HSCs to achieve hematopoietic compartment
reconstitution in a subject in need thereof; and b) administering a
second composition containing a MYC-composition, a
Bcl-2-composition, or both to the subject, where administering the
second composition achieves an at least 50% acceleration in
hematopoietic compartment reconstitution compared to hematopoietic
compartment reconstitution in a subject that is not administered
the second composition.
[0021] Other aspects of the present disclosure relate to a method
of enhancing hematopoietic compartment autoreconstitution in a
subject, by: administering a therapeutically effective amount of a
composition containing a MYC-composition, a Bcl-2-composition, or
both to a subject in need of hematopoietic compartment
autoreconstitution, where the MYC-composition, the
Bcl-2-composition, or both induces endogenous hematopoietic stem
cells (HSCs) to autoreconstitute the hematopoietic compartment in
the subject, and where hematopoietic compartment autoreconstitution
is enhanced compared to hematopoietic compartment
autoreconstitution in a subject that is not administered the
composition.
[0022] Other aspects of the present disclosure relate to a method
of treating a decrease in hematopoietic compartment cells due to
chemotherapy, by: administering a therapeutically effective amount
of a composition containing a MYC-composition, a Bcl-2-composition,
or both to a subject that is undergoing or has undergone
chemotherapy, where the MYC-composition, Bcl-2-composition, or both
induces endogenous hematopoietic stem cells (HSCs) to
autoreconstitute the hematopoietic compartment in the subject.
[0023] Other aspects of the present disclosure relate to a method
of treating a decrease in hematopoietic compartment cells due to
radiation therapy, by: administering a therapeutically effective
amount of a composition containing a MYC-composition, a
Bcl-2-composition, or both to a subject that is undergoing or has
undergone radiation therapy, where the MYC-composition,
Bcl-2-composition, or both induces endogenous hematopoietic stem
cells (HSCs) to autoreconstitute the hematopoietic compartment in
the subject.
[0024] Other aspects of the present disclosure relate to a method
of treating a bone marrow failure syndrome, by: administering a
therapeutically effective amount of a composition containing a
MYC-composition, a Bcl-2-composition, or both to a subject having a
bone marrow failure syndrome, where the MYC-composition,
Bcl-2-composition, or both induces endogenous hematopoietic stem
cells (HSCs) to autoreconstitute the hematopoietic compartment in
the subject.
[0025] Other aspects of the present disclosure relate to a use of a
composition containing a MYC-composition, a Bcl-2-composition, or
both in a subject that is in need or was in need of hematopoietic
compartment cell formation to enhance hematopoietic compartment
cell formation, where use of the composition enhances hematopoietic
compartment formation compared to hematopoietic compartment
formation in a patient that does not receive the composition.
[0026] In certain embodiments that may be combined with any of the
preceding embodiments, the subject is in need or was in need of
hematopoietic stem cell (HSC) transplantation. In certain
embodiments, the subject has or had a hematological malignancy, a
myeloma, multiple myeloma, a leukemia, acute lymphoblastic
leukemia, chronic lymphocytic leukemia, a lymphoma, indolent
lymphoma, non-Hodgkin lymphoma, diffuse B cell lymphoma, follicular
lymphoma, mantle cell lymphoma, T cell lymphoma, Hodgkin lymphoma,
a neuroblastoma, a retinoblastoma, Shwachman Diamond syndrome, a
brain tumor, Ewing's Sarcoma, a Desmoplastic small round cell
tumor, a relapsed germ cell tumor, a hematological disorder, a
hemoglobinopathy, an autoimmune disorder, juvenile idiopathic
arthritis, systemic lupus erythematosus, severe combined
immunodeficiency, congenital neutropenia with defective stem cells,
severe aplastic anemia, a sickle-cell disease, a myelodysplastic
syndrome, chronic granulomatous disease, a metabolic disorder,
Hurler syndrome, Gaucher disease, osteopetrosis, malignant
infantile osteopetrosis, heart disease, HIV, or AIDS. In certain
embodiments, the subject has had an organ transplant. In certain
embodiments that may be combined with any of the preceding
embodiments, the subject is undergoing or underwent and HSC
transplant. In certain embodiments, the composition containing a
MYC-composition, a Bcl-2-composition, or both is administered
before, after, or concurrently with the HSC transplant. In certain
embodiments, the composition containing a MYC-composition, a
Bcl-2-composition, or both is administered at least 5 days, at
least 4 days, at least 3 days, at least 2 days, or at least 1 day
before HSC transplantation. In certain embodiments, the composition
containing a MYC-composition, a Bcl-2-composition, or both is
administered concurrently with HSC transplantation. In certain
embodiments, the composition containing a MYC-composition, a
Bcl-2-composition, or both is administered at least 1 day, at least
2 days, at least three days, at least 4 days, at least 5 days, at
least 6 days, at least 1 week, at least 2 weeks, or at least 3
weeks after the HSC transplantation. In certain embodiments that
may be combined with any of the preceding embodiments,
administration of the composition containing a MYC-composition, a
Bcl-2-composition, or both results in an expansion of the
transplanted HSCs. In certain embodiments that may be combined with
any of the preceding embodiments, the transplanted HSCs were
cultured in the presence of a MYC-composition, a Bcl-2-composition,
or both prior to HSC transplantation. In certain embodiments,
culturing the HSCs in the presence of the MYC-composition,
Bcl-2-composition, or both conditionally immortalized the HSCs. In
certain embodiments, immortalization of the HSCs resulted in an
expansion of the HSCs. In certain embodiments that may be combined
with any of the preceding embodiments, the transplanted HSCs
included the MYC-composition, Bcl-2-composition, or both as a
result of being cultured in the presence of the MYC-composition,
Bcl-2-composition, or both. In certain embodiments that may be
combined with any of the preceding embodiments, the transplanted
HSCs were obtained from bone marrow, from peripheral blood cells,
from peripheral blood cells that have undergone apheresis, from
peripheral blood cells that have undergone leukapheresis, from
umbilical cord blood, from amniotic fluid, from cultured HSC cells,
from an immortalized HSC cell line, or from a conditionally
immortalized HSC cell line. In certain embodiments that may be
combined with any of the preceding embodiments, the HSC
transplantation procedure is or was a myeloablative HSC
transplantation procedure. In certain embodiments that may be
combined with any of the preceding embodiments, the HSC
transplantation procedure is or was a non-myeloablative HSC
transplantation procedure. In certain embodiments that may be
combined with any of the preceding embodiments, the HSC
transplantation is or was an autologous HSC transplantation. In
certain embodiments that may be combined with any of the preceding
embodiments, the HSC transplantation is or was an allogenic HSC
transplantation. In certain embodiments that may be combined with
any of the preceding embodiments, administration of the composition
containing a MYC-composition, a Bcl-2-composition, or both
accelerates hematopoietic compartment reconstitution after HSC
transplantation in the subject. In certain embodiments,
administering the composition containing a MYC-composition, a
Bcl-2-composition, or both achieves an at least 50% acceleration in
hematopoietic compartment reconstitution compared to hematopoietic
compartment reconstitution in a subject that is not administered
the composition. In certain embodiments that may be combined with
any of the preceding embodiments, the accelerated hematopoietic
compartment reconstitution in the subject results in T cell
compartment reconstitution that is accelerated by at least 50%
compared to T cell reconstitution in a subject that is not
administered the composition. In certain embodiments that may be
combined with any of the preceding embodiments, the accelerated
hematopoietic compartment reconstitution in the subject results in
B cell compartment reconstitution that is accelerated by at least
50% compared to B cell reconstitution in a subject that is not
administered the composition. In certain embodiments that may be
combined with any of the preceding embodiments, the accelerated
hematopoietic compartment reconstitution in the subject results in
NK-cell compartment reconstitution that is accelerated by at least
50% compared to NK-cell reconstitution in a subject that is not
administered the composition. In certain embodiments that may be
combined with any of the preceding embodiments, the accelerated
hematopoietic compartment reconstitution in the subject results in
myeloid cell compartment reconstitution that is accelerated by at
least 50% compared to myeloid cell compartment reconstitution in a
subject that is not administered the composition. In certain
embodiments that may be combined with any of the preceding
embodiments, the accelerated hematopoietic compartment
reconstitution in the subject results in neutrophil recovery that
is accelerated by at least 50% compared to neutrophil recovery in a
subject that is not administered the composition. In certain
embodiments that may be combined with any of the preceding
embodiments, administration of the composition containing a
MYC-composition, a Bcl-2-composition, or both results in a 50%
increase in HSC productive homing to bone marrow niches in the
subject. In certain embodiments that may be combined with any of
the preceding embodiments, further including administering a second
composition containing at least one cytokine, growth factor,
antibody, and/or small molecule modifier. In certain embodiments,
the third composition further contains a pharmaceutically
acceptable carrier. In certain embodiments that may be combined
with any of the preceding embodiments, the transplanted HSCs are
human HSCs. In certain embodiments, the subject is in need or was
in need of hematopoietic compartment autoreconstitution. In certain
embodiments, the MYC-composition, Bcl-2-composition, or both
induces endogenous hematopoietic stem cells (HSCs) to
autoreconstitute the hematopoietic compartment in the subject. In
certain embodiments that may be combined with any of the preceding
embodiments, the hematopoietic compartment autoreconstitution is
enhanced compared to hematopoietic compartment autoreconstitution
in a subject that is not administered the composition containing a
MYC-composition, a Bcl-2-composition, or both. In certain
embodiments that may be combined with any of the preceding
embodiments, the enhanced hematopoietic compartment
autoreconstitution in the subject results in enhanced T cell
compartment autoreconstitution, enhanced B cell compartment
autoreconstitution, enhanced NK-cell compartment
autoreconstitution, enhanced myeloid cell compartment
autoreconstitution, or neutrophil recovery. In certain embodiments
that may be combined with any of the preceding embodiments,
administration of the composition containing a MYC-composition, a
Bcl-2-composition, or both results in an expansion of endogenous
HSCs. In certain embodiments that may be combined with any of the
preceding embodiments, the subject is undergoing or has undergone
chemotherapy. In certain embodiments, administration of the
composition containing a MYC-composition, a Bcl-2-composition, or
both prevents a decrease in hematopoietic compartment cells due to
the chemotherapy. In certain embodiments that may be combined with
any of the preceding embodiments, administration of the composition
containing a MYC-composition, a Bel-2-composition, or both prevents
a decrease in the amount of endogenous HSCs due to the
chemotherapy. In certain embodiments that may be combined with any
of the preceding embodiments, the subject is undergoing or has
undergone radiation therapy. In certain embodiments, administration
of the composition containing a MYC-composition, a
Bcl-2-composition, or both prevents a decrease in hematopoietic
compartment cells due to the radiation therapy. In certain
embodiments that may be combined with any of the preceding
embodiments, administration of the composition containing a
MYC-composition, a Bcl-2-composition, or both prevents a decrease
in the amount of endogenous HSCs due to the radiation therapy. In
certain embodiments that may be combined with any of the preceding
embodiments, the subject has a bone marrow failure syndrome. In
certain embodiments, the bone marrow failure syndrome is aplastic
anemia or Gulf War Syndrome. In certain embodiments, the bone
marrow failure syndrome is an inherited bone marrow failure
syndrome (IBMFS). In certain embodiments, the IBMFS is selected
from amegakaryocytic thrombocytopenia, Diamond-Blackfan anemia,
dyskeratosis congenita, fanconi anemia, Pearson syndrome, severe
congenital neutropenia, Shwachman-Diamond syndrome, and
thrombocytopenia absent radii, IVIC syndrome, WT syndrome,
radio-ulnar synostosis, and ataxia pancytopenia. In certain
embodiments that may be combined with any of the preceding
embodiments, administration of the composition containing a
MYC-composition, a Bcl-2-composition, or both prevents a decrease
in hematopoietic compartment cells due to the bone marrow failure
syndrome. In certain embodiments that may be combined with any of
the preceding embodiments, administration of the composition
containing a MYC-composition, a Bcl-2-composition, or both prevents
a decrease in the amount of endogenous HSCs due to the bone marrow
failure syndrome. In certain embodiments that may be combined with
any of the preceding embodiments, the MYC-composition is TAT-MYC.
In certain embodiments that may be combined with any of the
preceding embodiments, the therapeutically effective amount of the
composition containing a MYC-composition is at least 0.1 mg/kg, at
least 0.2 mg/kg, at least 0.3 mg/kg, at least 0.4 mg/kg, at least
0.5 mg/kg, at least 0.6 mg/kg, at least 0.7 mg/kg, at least 0.8
mg/kg, at least 0.9 mg/kg, at least 1 mg/kg, at least 2 mg/kg, at
least 3 mg/kg, at least 4 mg/kg, at least 5 mg/kg, at least 6
mg/kg, at least 7 mg/kg, at least 8 mg/kg, at least 9 mg/kg, at
least 10 mg/kg, at least 20 mg/kg, at least 30 mg/kg, at least 40
mg/kg, or at least 50 mg/kg of the subject's weight. In certain
embodiments that may be combined with any of the preceding
embodiments, the Bcl-2-composition is TAT-Bcl-2.
[0027] In certain embodiments that may be combined with any of the
preceding embodiments, the therapeutically effective amount of the
composition comprising a Bcl-2-composition is at least 0.1 mg/kg,
at least 0.2 mg/kg, at least 0.3 mg/kg, at least 0.4 mg/kg, at
least 0.5 mg/kg, at least 0.6 mg/kg, at least 0.7 mg/kg, at least
0.8 mg/kg, at least 0.9 mg/kg, at least 1 mg/kg, at least 2 mg/kg,
at least 3 mg/kg, at least 4 mg/kg, at least 5 mg/kg, at least 6
mg/kg, at least 7 mg/kg, at least 8 mg/kg, at least 9 mg/kg, at
least 10 mg/kg, at least 20 mg/kg, at least 30 mg/kg, at least 40
mg/kg, or at least 50 mg/kg of the subject's weight. In certain
embodiments that may be combined with any of the preceding
embodiments, the composition containing a MYC-composition, a
Bcl-2-composition, or both further contains a pharmaceutically
acceptable carrier.
[0028] In certain embodiments that may be combined with any of the
preceding embodiments, the composition containing a
MYC-composition, the subject is a human patient. In certain
embodiments that may be combined with any of the preceding
embodiments, the subject is a non-human animal.
[0029] Other aspects of the present disclosure relate to a use of a
composition containing a MYC-composition, a Bcl-2-composition, or
both in a patient who has had or will receive a hematopoietic stem
cell (HSC) transplant to accelerate hematopoietic compartment
reconstitution, where use of the composition achieves an at least
50% acceleration in hematopoietic compartment reconstitution in the
patient compared to hematopoietic compartment reconstitution in a
subject that does not receive the composition.
[0030] Other aspects of the present disclosure relate to a use of a
composition containing a MYC-composition, a Bcl-2-composition, or
both in a patient who has or has had a decrease in hematopoietic
compartment cells to enhance hematopoietic compartment
autoreconstitution, where the MYC-composition, Bcl-2-composition,
or both induces endogenous hematopoietic stem cells (HSCs) to
autoreconstitute the hematopoietic compartment in the patient, and
where use of the composition enhances hematopoietic compartment
autoreconstitution compared to hematopoietic compartment
autoreconstitution in a patient that does not receive the
composition.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIGS. 1A-1D depict the results of FACS staining showing the
development of T-cells in mice 4 weeks after transplant with
expanded bone marrow cells and treatment with TAT-MYC. The panels
show flow cytometry of TCR.beta. positive PBMCs. FIG. 1A and FIG.
1B depict flow cytometry of cells from Rag-1.sup.-/- (FIG. 1A) and
C57BL/6 (FIG. 1B) control mice that did not receive a cell
transplant or treatment with TAT-MYC. FIG. 1C and FIG. 1D depict
flow cytometry of cells from Rag-1.sup.-/- mice injected with
5.times.10.sup.3 expanded bone marrow cells alone (FIG. 1C), or
also receiving a 10 .mu.g injection Tat-MYC 24 hours
post-transplant (FIG. 1D).
[0032] FIGS. 2A-2D depict the results of FACS staining showing the
development of B-cells in mice 4 weeks after transplant with
expanded bone marrow cells and treatment with TAT-MYC. The panels
show flow cytometry of B220 positive PBMCs. FIG. 2A and FIG. 2B
depict flow cytometry of cells from Rag-1.sup.-/- (FIG. 2A) and
C57BL/6 (FIG. 2B) control mice that did not receive a cell
transplant or treatment with TAT-MYC. FIG. 2C and FIG. 2D depict
flow cytometry of cells from Rag-1.sup.-/- mice injected with
5.times.10.sup.3 expanded bone marrow cells alone (FIG. 2C), or
also receiving a 10 .mu.g injection Tat-MYC 24 hours
post-transplant (FIG. 2D).
[0033] FIGS. 3A-3D depict the results of FACS staining showing the
development of T-cells in mice 8 weeks after transplant with
expanded bone marrow cells and treatment with TAT-MYC. The panels
show flow cytometry of TCR.beta. positive PBMCs. FIG. 3A and FIG.
3B depict flow cytometry of cells from Rag-1.sup.-/- (FIG. 3A) and
C57BL/6 (FIG. 3B) control mice that did not receive a cell
transplant or treatment with TAT-MYC. FIG. 3C and FIG. 3D depict
flow cytometry of cells from Rag-1.sup.-/- mice injected with
5.times.10.sup.3 expanded bone marrow cells alone (FIG. 3C), or
also receiving a 10 .mu.g injection Tat-MYC 24 hours
post-transplant (FIG. 3D).
[0034] FIGS. 4A-4D depict the results of FACS staining showing the
development of B-cells in mice 8 weeks after transplant with
expanded bone marrow cells and treatment with TAT-MYC. The panels
show flow cytometry of B220 positive PBMCs. FIG. 4A and FIG. 4B
depict flow cytometry of cells from Rag-1.sup.-/- (FIG. 4A) and
C57BL/6 (FIG. 4B) control mice that did not receive a cell
transplant or treatment with TAT-MYC. FIG. 4C and FIG. 4D depict
flow cytometry of cells from Rag-1.sup.-/- mice injected with
5.times.10.sup.3 expanded bone marrow cells alone (FIG. 4C), or
also receiving a 10 .mu.g injection Tat-MYC 24 hours
post-transplant (FIG. 4D).
[0035] FIGS. 5A-5D depict the results of FACS staining showing the
development of T-cells in mice 4 weeks after transplant with
freshly isolated whole bone marrow cells and treatment with
TAT-MYC. The panels show flow cytometry of isolated PBMCs gated for
CD8.times.CD4 positive cells. FIG. 5A and FIG. 5B depict flow
cytometry of cells from Rag-1.sup.-/- (FIG. 5A) and C57BL/6 (FIG.
5B) control mice that did not receive a cell transplant or
treatment with TAT-MYC. FIG. 5C and FIG. 5D depict flow cytometry
of cells from Rag-1.sup.-/- mice injected with 1.times.10.sup.6
whole bone marrow cells alone (FIG. 5C), or also receiving a 10
.mu.g injection Tat-MYC 24 hours post-transplant (FIG. 5D).
[0036] FIGS. 6A and 6B graphically depict the percentage of T cells
for the full cohort of mice shown in FIG. 5. FIG. 6A shows the %
CD4 positive T cells in C57BL/6 control mice (untreated; first
column), Rag-1.sup.-/- mice treated with 10.sup.6 BM cells only
(middle column); and Rag-1.sup.-/- mice treated with 10.sup.6 BM
cells followed 24 hours later with 10 .mu.g TAT-MYC (last column).
FIG. 6B shows the % CD8 T cells in C57BL/6 control mice (untreated;
first column), Rag-1.sup.-/- mice treated with 10.sup.6 BM cells
only (middle column); and Rag-1.sup.-/- mice treated with 10.sup.6
BM cells followed 24 hours later with 10 .mu.g TAT-MYC (last
column).
[0037] FIGS. 7A-7D depict the results of FACS staining showing the
development of B-cells in mice 4 weeks after transplant with
freshly isolated whole bone marrow cells and treatment with
TAT-MYC. The panels show flow cytometry of isolated PBMCs gated for
B220.times.CD19 positive cells. FIG. 7A and FIG. 7B depict flow
cytometry of cells from Rag-1.sup.-/- (FIG. 7A) and C57BL/6 (FIG.
7B) control mice that did not receive a cell transplant or
treatment with TAT-MYC. FIG. 7C and FIG. 7D depict flow cytometry
of cells from Rag-1.sup.-/- mice injected with 1.times.10.sup.6
whole bone marrow cells alone (FIG. 7C), or also receiving a 10
.mu.g injection Tat-MYC 24 hours post-transplant (FIG. 7D).
[0038] FIG. 8 graphically depicts the percentage of B cells for the
full cohort of mice shown in FIG. 7. The graph shows the %
CD19.times.B220 positive B cells in C57BL/6 control mice
(untreated; first column), Rag-1.sup.-/- mice treated with 10.sup.6
BM cells only (middle column); and Rag-1.sup.-/- mice treated with
10.sup.6 BM cells followed 24 hours later with 10 .mu.g TAT-MYC
(last column).
[0039] FIGS. 9A-9D graphically depict activation of splenic T-cells
and B-cells from Rag-1.sup.-/- mice cohorts shown in FIGS. 5-8.
FIG. 9A and FIG. 9B show the activation of T cells (FIG. 9A) and B
cells (FIG. 9B) from Rag-1.sup.-/- mice transplanted with 10.sup.6
fresh bone marrow cells, but not treated with TAT-MYC. FIG. 9C and
FIG. 9D show the activation of T cells (FIG. 9C) and B cells (FIG.
9D) from Rag-1.sup.-/- mice treated with 10 .mu.g TAT-MYC following
transplantation with fresh bone marrow cells.
[0040] FIGS. 10A-10C depict the results of FACS staining showing
the reconstitution of T-cells in mice 2 weeks after non-lethal
challenge with 5FU followed by treatment with TAT-MYC. The panels
show flow cytometry of isolated PBMCs gated for CD8.times.CD4
positive cells. FIG. 10A depicts flow cytometry of cells from
C57BL/6 control mice that did not receive treatment with TAT-MYC.
FIG. 10B depicts flow cytometry of cells from C57BL/6 mice treated
with 10 .mu.g TAT-CRE. FIG. 10C depicts flow cytometry of cells
from C57BL/6 mice treated with 10 .mu.g Tat-MYC 24 hours post-5FU
challenge.
[0041] FIG. 11 graphically depicts the percentage of reconstituted
CD4 T cells for the full cohort of 5FU challenged mice shown in
FIG. 10. The graph shows the % CD4 positive T cells in C57BL/6
control mice (untreated; first column), C57BL/6 mice treated with
10 .mu.g TAT-MYC (middle column); and C57BL/6 mice treated with 10
.mu.g TAT-CRE (last column).
[0042] FIG. 12 graphically depicts the percentage of reconstituted
CD8 T cells for the full cohort of 5FU challenged mice shown in
FIG. 10. The graph shows the % CD8 positive T cells in C57BL/6
control mice (untreated; first column), C57BL/6 mice treated with
10 .mu.g TAT-MYC (middle column); and C57BL/6 mice treated with 10
.mu.g TAT-CRE (last column).
[0043] FIGS. 13A-13E depict the results of FACS staining showing
the reconstitution of T-cells in mice 4 weeks after sub-lethal
irradiation followed transplantation of freshly-isolated whole bone
marrow and treatment with TAT-MYC. The panels show flow cytometry
of isolated PBMCs gated for CD4.times.TCR.beta. positive cells.
FIG. 13A and FIG. 13B depict flow cytometry of cells from
Rag-1.sup.-/- (FIG. 13A) and C57BL/6 (FIG. 13B) control mice that
did not receive a cell transplant or treatment with TAT-MYC. FIG.
13C, FIG. 13D, and FIG. 13E depict flow cytometry of cells from
Rag-1.sup.-/- mice injected with 1.times.10.sup.6 whole bone marrow
cells alone (FIG. 13C), or also receiving an intravenous (FIG. 13D)
or intramuscular (FIG. 13E) injection of 10 .mu.g Tat-MYC 24 hours
post-transplant.
[0044] FIG. 14 graphically depicts the percentage of reconstituted
CD4 T cells for the full cohort of 5FU challenged mice shown in
FIG. 13. The graph shows the % CD4 positive T cells in
Rag-1.sup.-/- control mice (untreated; first column), Rag-1.sup.-/-
mice treated with 10 .mu.g TAT-MYC intravenously (middle column);
and Rag-1.sup.-/- mice treated with 10 .mu.g TAT-CRE
intramuscularly (last column).
[0045] FIGS. 15A-15E depict the results of FACS staining showing
the reconstitution of B-cells in mice 4 weeks after sub-lethal
irradiation followed by transplantation of freshly isolated whole
bone marrow and treatment with TAT-MYC. The panels show flow
cytometry of isolated PBMCs gated for IgM.times.CD19 positive
cells. FIG. 15A and FIG. 15B depict flow cytometry of cells from
Rag-1.sup.-/- (FIG. 15A) and C57BL/6 (FIG. 15B) control mice that
did not receive a cell transplant or treatment with TAT-MYC. FIG.
15C, FIG. 15D, and FIG. 15E depict flow cytometry of cells from
Rag-1.sup.-/- mice injected with 1.times.10.sup.6 whole bone marrow
cells alone (FIG. 15C), or also receiving an intravenous (FIG. 15D)
or intramuscular (FIG. 15E) injection of 10 .mu.g Tat-MYC 24 hours
post-transplant.
[0046] FIG. 16 graphically depicts the percentage of reconstituted
B cells for the full cohort of sublethally irradiated mice that
were given HSC transplants and then treated with either Tat-MYC or
Tat-Cre, or a control 24 hours after the transplant shown in FIG.
15. The graph shows the % CD19.times.B220 positive B cells in
Rag-1.sup.-/- control mice (untreated; first column), Rag-1.sup.-/-
mice treated with 10 .mu.g TAT-MYC intravenously (middle column);
and Rag-1.sup.-/- mice treated with 10 .mu.g TAT-CRE
intramuscularly (last column).
[0047] FIGS. 17A-17G depict shows a functional analysis of human
cord blood derived protein-transduced long term (ptlt)-HSC in vivo.
FIG. 17A depicts the results of FACS analysis showing
reconstitution of the bone marrow of cohorts of sublethally
irradiated NSG mice given transplants of 10.sup.6 cord blood cells
expanded in vitro in a cocktail of cytokines (first panel; FCB), or
expanded in a cocktail of cytokines supplemented with Tat-Myc and
Tat-Bcl-2 (second panel; FCB TMTB), or 5.times.10.sup.6 fresh
un-manipulated cord blood cells (third panel; Fresh FCB). FIG. 17B
depicts the results of FACS analysis of bone marrow, spleen and
thymus cells from the xenochimaeric mice reconstituted with
ptlt-HSC shown in the second panel of FIG. 17A. All cells were
stained for human CD45. Gating on CD45+ cells showed human
CD34+CD38lo cells in the bone marrow (first panel; BM); human CD19+
and human CD3+ lymphocytes in the spleen (second panel; spleen);
and human CD3+ cells in the thymus (third panel; thymus). FIG. 17C
depicts the results of FACS analysis of human splenic B cells that
developed in the NSG mouse shown in the second panel of FIG. 17A.
The splenic B cells were labeled with CFSE and cultured in the
presence of monoclonal antibodies to human CD40 and IgM. Human B
cells from this mouse underwent proliferation following stimulation
of their antigen receptor. FIG. 17D shows a graphical
representation of the quantification of myeloerythroid colonies
(Burst Forming Unit Erythroid (BFU-E), Colony Forming Units
Megakaryocyte (CFU-M), Colony Forming Units Granulocyte (CFU-G),
and Colony Forming Units Granulocyte Monocyte (CFU-GM)) from human
CD34+CD38.sup.lo cells after plating on methycellulose. The human
CD34+CD38.sup.lo cells were obtained from the bone marrow of the
NSG xenochimaeric mouse shown in FIG. 17A second panel further
analyzed in FIG. 17B first panel. FIG. 17E shows a graphical
representation of the quantification of the development of
myeloerythroid colonies following replating on methycellulose. FIG.
17F shows a graphical representation of the quantification of
myeloid and lymphoid cell differentiation (CD11b, CD33, CD3, and
CD19 expression) in the CD45 positive population of bone marrow
cells from the NSG mouse shown in FIG. 17A second panel. FIG. 17G
shows a graphical representation of the quantification of myeloid
and lymphoid cell differentiation (CD11b, CD33, CD3, and CD19
expression) in the CD45 positive population of spleen cells from
the NSG mouse shown in FIG. 17A second panel.
[0048] FIGS. 18A-18F depict the results of FACS staining showing
the reconstitution of the peripheral blood in NSG mice 8 weeks
after sub-lethal irradiation followed by transplantation of fresh
fetal cord blood cells and treatment with TAT-MYC or Tat-Cre
control protein. The panels show flow cytometry of isolated PBMCs
gated for human CD45 positive cells. FIGS. 18A-18C depict flow
cytometry of cells from NSG mice that received 5.times.10.sup.5
(FIG. 18A), 1.times.10.sup.6 (FIG. 18B), or 5.times.10.sup.6 (FIG.
18C) freshly isolated fetal cord blood cells followed 24 hours
later by an injection of 10 Tat-Cre. FIGS. 18D-18F depict flow
cytometry of cells from NSG mice that received 5.times.10.sup.5
(FIG. 18D), 1.times.10.sup.6 (FIG. 18E), or 5.times.10.sup.6 (FIG.
18F) freshly isolated fetal cord blood cells followed 24 hours
later by an injection of 10 .mu.g Tat-MYC.
[0049] FIGS. 19A-19F depict the results of FACS staining showing
the reconstitution of the bone marrow from NSG mice 8 weeks after
sub-lethal irradiation followed transplantation of fresh fetal cord
blood cells and treatment with TAT-MYC or Tat-Cre control protein.
The panels show flow cytometry of isolated bone marrow gated for
human CD45 positive cells. FIGS. 19A-19C depict flow cytometry of
cells from NSG mice that received 5.times.10.sup.5 (FIG. 19A),
1.times.10.sup.6 (FIG. 19B), or 5.times.10.sup.6 (FIG. 19C) freshly
isolated fetal cord blood cells followed 24 hours later by an
injection of 10 .mu.g Tat-Cre. FIGS. 19D-19F depict flow cytometry
of cells from NSG mice that received 5.times.10.sup.5 (FIG. 19D),
1.times.10.sup.6 (FIG. 19E), or 5.times.10.sup.6 (FIG. 19F) freshly
isolated fetal cord blood cells followed 24 hours later by an
injection of 10 .mu.g Tat-MYC.
[0050] FIGS. 20A-20F depict the results of FACS staining showing
the reconstitution of the spleen in NSG mice 8 weeks after
sub-lethal irradiation followed transplantation of fresh fetal cord
blood cells and treatment with TAT-MYC or Tat-Cre control protein.
The panels show flow cytometry of isolated spleen cells gated for
human CD45 positive cells. FIGS. 20A-20C depict flow cytometry of
cells from NSG mice that received 5.times.10.sup.5 (FIG. 20A),
1.times.10.sup.6 (FIG. 20B), or 5.times.10.sup.6 (FIG. 20C) freshly
isolated fetal cord blood cells followed 24 hours later by an
injection of 10 .mu.g Tat-Cre. FIGS. 20D-20F depict flow cytometry
of cells from NSG mice that received 5.times.10.sup.5 (FIG. 20D),
1.times.10.sup.6 (FIG. 20E), or 5.times.10.sup.6 (FIG. 20F) freshly
isolated fetal cord blood cells followed 24 hours later by an
injection of 10 .mu.g Tat-MYC.
[0051] FIGS. 21A and 21B depict the results of FACS staining
showing the reconstitution of the peripheral blood in NSG mice 8
weeks after sub-lethal irradiation followed transplantation of
fresh fetal cord blood cells that were treated in vitro for 1 hour
with TAT-MYC prior to injecting into mice. The panels show flow
cytometry of isolated PBMCs gated for human CD45 positive cells.
FIG. 21A depicts flow cytometry of cells from NSG mice that
received 5.times.10.sup.6 freshly isolated fetal cord blood cells.
FIG. 21B depicts flow cytometry of cells from NSG mice that
received 5.times.10.sup.6 freshly isolated fetal cord blood cells
treated in vitro with Tat-MYC for 1 hour prior to injecting the
cells into mice.
[0052] FIGS. 22A-22F depict the results of FACS staining showing
the long-term reconstitution of the bone marrow, spleen and thymus
in NSG mice 8 months after sub-lethal irradiation followed
transplantation of fresh fetal cord blood cells that were treated
in vitro for 1 hour with TAT-MYC prior to injecting into mice. The
panels show flow cytometry of isolated bone marrow, spleen and
thymus cells gated for human CD45 positive cells. FIG. 22A depicts
flow cytometry of bone marrow cells from NSG mice that received
5.times.10.sup.6 freshly isolated fetal cord blood cells. FIG. 22B
depicts flow cytometry of bone marrow cells from NSG mice that
received 5.times.10.sup.6 freshly isolated fetal cord blood cells
treated in vitro with Tat-MYC for 1 hour prior to injecting the
cells into mice. FIG. 22C depicts flow cytometry of spleen cells
from NSG mice that received 5.times.10.sup.6 freshly isolated fetal
cord blood cells. FIG. 22D depicts flow cytometry of Spleen cells
from NSG mice that received 5.times.10.sup.6 freshly isolated fetal
cord blood cells treated in vitro with Tat-MYC for 1 hour prior to
injecting the cells into mice. FIG. 22E depicts flow cytometry of
thymus cells from NSG mice that received 5.times.10.sup.6 freshly
isolated fetal cord blood cells. FIG. 22F depicts flow cytometry of
thymus cells from NSG mice that received 5.times.10.sup.6 freshly
isolated fetal cord blood cells treated in vitro with Tat-MYC for 1
hour prior to injecting the cells into mice.
[0053] FIGS. 23A-23C depict a FACS analysis of the peripheral blood
from a control NSG mouse (FIG. 23A), a sublethally irradiated NSG
mouse given transplants of 5.times.10.sup.6 C-GSF mobilized adult
blood cells expanded in vitro in a cocktail of cytokines (FIG.
23B), or 5.times.10.sup.6 C-GSF mobilized adult blood cells
expanded in a cocktail of cytokines supplemented with Tat-MYC and
Tat-Bcl-2 (FIG. 23C).
[0054] FIG. 24 depicts the amino acid and nucleic acid sequences
for some embodiments of the Tat-Myc polypeptide. Figure discloses
"6 Histidine tag" as SEQ ID NO: 6.
[0055] FIG. 25 depicts the amino acid and nucleic acid sequences
for some embodiments of the Bcl-2 domain polypeptide. Figure
discloses "6 Histidine tag" as SEQ ID NO: 6.
DETAILED DESCRIPTION
[0056] The present disclosure relates, in part, to enhancing
hematopoietic compartment cell formation in a subject in need
thereof by administering a composition containing a
MYC-composition, such as a protein transduction domain-MYC
(PTD-MYC) fusion protein, a composition containing a
Bcl-2-composition, such as a protein transduction domain-Bcl-2
(PTD-Bcl-2) fusion protein, or both. In certain aspects, the
present disclosure relates to accelerating hematopoietic stem cell
(HSC) engraftment and hematopoietic compartment reconstitution in
an HSC transplant recipient by administering a composition
containing a MYC-composition, such as a protein transduction
domain-MYC (PTD-MYC) fusion protein, a composition containing a
Bcl-2-composition, such as a protein transduction domain-Bcl-2
(PTD-Bcl-2) fusion protein, or both. In some embodiments, the HSC
transplant recipient is also administered a composition containing
a MYC-composition, such as a protein transduction domain-MYC
(PTD-MYC) fusion protein, a composition containing a
Bcl-2-composition, such as a protein transduction domain-Bcl-2
(PTD-Bcl-2) fusion protein, or both to maintain and/or further
accelerate HSC engraftment and hematopoietic compartment
reconstitution in the HSC transplant recipient. As used herein, HSC
transplantation includes, without limitation, bone marrow
transplantation.
[0057] Moreover, the present disclosure is based, at least in part,
on the discovery that administering a composition having a fusion
protein containing a MYC polypeptide and a PTD, such as the HIV TAT
protein transduction domain, after bone transplantation reduced the
time required for recovery of mature hematopoietic lineages after
bone marrow transplantation. For example, it was shown that the
time required for T-cell recovery was reduced from about 9 weeks to
about 4 weeks, and the time required for B-cell recovery was
reduced from about 10 weeks to about 4 weeks in lethally irradiated
mice that were administered a MYC-composition after bone marrow
transplantation (Example 1). Accordingly, one aspect of the present
disclosure provides methods of accelerating hematopoietic
compartment reconstitution after hematopoietic stem cell (HSC)
transplantation in a subject, by: a) administering a
therapeutically effective amount of a first composition containing
HSCs to achieve hematopoietic compartment reconstitution in a
subject in need thereof; and b) administering a second composition
containing a MYC-composition, a Bcl-2-composition, or both to the
subject, where administering the second composition achieves an at
least 50% acceleration in hematopoietic compartment reconstitution
compared to hematopoietic compartment reconstitution in a subject
that is not administered the second composition.
[0058] In other aspects, the present disclosure relates to
enhancing hematopoietic compartment autoreconstitution in a subject
in need thereof by administering a composition having a protein
transduction domain-MYC (PTD-MYC) fusion protein. The present
disclosure is also based, at least in part, on the novel discovery
that administration of a MYC-composition can enhance recovery in a
subject undergoing chemotherapy and/or radiation therapy after
suffering a decrease hematopoietic compartment cells due to the
therapy. For example, it was shown that administration of a
MYC-composition to a subject after treatment with the
chemotherapeutic drug 5-fluorouracil accelerated recovery of
hematopoietic compartment cells (e.g., CD8+ T-cells) following the
reduction due to the 5-fluorouracil treatment (FIG. 14).
[0059] Another aspect of the present disclosure provides methods of
enhancing hematopoietic compartment autoreconstitution in a
subject, by: administering a therapeutically effective amount of a
composition containing a MYC-composition, a composition containing
a Bcl-2-composition, or both to a subject in need of hematopoietic
compartment autoreconstitution, where the MYC-composition, the
Bcl-2-composition, or both induces endogenous HSCs to
autoreconstitute the hematopoietic compartment in the subject, and
where hematopoietic compartment autoreconstitution is enhanced
compared to hematopoietic compartment autoreconstitution in a
subject that is not administered the composition.
[0060] In other aspects, the present disclosure relates, in part,
to enhancing hematopoietic compartment reconstitution in a subject
in need of hematopoietic stem cell transplantation by pre-treating
a population of HSCs with a MYC-composition, such as a protein
transduction domain-MYC (PTD-MYC) fusion protein; a Bcl-2
composition, such as a PTD-Bcl-2 fusion protein; or both, prior to
administering the HSCs to the subject. In certain aspects, the
present disclosure relates to accelerating hematopoietic stem cell
(HSC) engraftment and hematopoietic compartment reconstitution in
an HSC transplant recipient by pre-treating HSCs with a protein
transduction domain-MYC (PTD-MYC) fusion protein, a protein
transduction domain-Bcl-2 (PTD-Bcl-2) fusion protein, or both prior
to administering the HSCs to the subject. Surprisingly,
pre-treating HSCs for at least as little as 1 hour prior to
transplantation (and perhaps as little as about 10 minutes) is
sufficient for the MYC-composition, the Bcl-2 composition, or both
to achieve an enhancement in hematopoietic compartment
reconstitution.
[0061] Accordingly, certain preferred embodiments relate to methods
of enhancing hematopoietic compartment reconstitution to a subject
in need of hematopoietic stem cell transplantation, by treating a
population of hematopoietic stem cells with a composition
containing a MYC-composition, a composition containing a
Bcl-2-composition, or both, for less than about 13 days; and
administering to the subject, a therapeutically effective amount of
the treated population of hematopoietic stem cells to reconstitute
the hematopoietic compartment of the subject, wherein hematopoietic
compartment reconstitution is enhanced compared to hematopoietic
compartment reconstitution in a subject that is administered a
population of hematopoietic stem cells that were not treated with
the composition containing a MYC-composition, the composition
containing a Bcl-2-composition, or both. In some embodiments, the
population of hematopoietic stem cells is treated with the
composition containing a MYC-composition, the composition
containing a Bcl-2-composition, or both, for less than about or
about 12 days to less than about or about 1 days, for example, less
than about or about 12 days, less than about or about 11 days, less
than about or about 10 days, less than about or about 9 days, less
than about or about 8 days, less than about or about 7 days, less
than about or about 6 days, less than about or about 5 days, less
than about or about 4 days, less than about or about 2 days, or
less than about or about 1 day. In some embodiments, the population
of hematopoietic stem cells is treated with the composition
containing a MYC-composition, the composition containing a
Bcl-2-composition, or both, for less than about or about 24 hours
to less than about or about 1 hour, for example, less than about or
about 24 hours, less than about or about 23 hours, less than about
or about 22 hours, less than about or about 21 hours, less than
about or about 20 hours, less than about or about 19 hours, less
than about or about 18 hours, less than about or about 17 hours,
less than about or about 16 hours, less than about or about 15
hours, less than about or about 14 hours, less than about or about
13 hours, less than about or about 12 hours, less than about or
about 11 hours, less than about or about 10 hours, less than about
or about 9 hours, less than about or about 8 hours, less than about
or about 7 hours, less than about or about 6 hours, less than about
or about 5 hours, less than about or about 4 hours, less than about
or about 3 hours, less than about or about 2 hours, or less than
about or about 1 hour. In some embodiments, the population of
hematopoietic stem cells is treated with the composition containing
a MYC-composition, the composition containing a Bcl-2-composition,
or both, for less than about or about 60 minutes to less than about
or about 10 minutes, for example, less than about or about 60
minutes, less than about or about 55 minutes, less than about or
about 50 minutes, less than about or about 45 minutes, less than
about or about 40 minutes, less than about or about 35 minutes,
less than about or about 30 minutes, less than about or about 29
minutes, less than about or about 28 minutes, less than about or
about 27 minutes, less than about or about 26 minutes, less than
about or about 25 minutes, less than about or about 24 minutes,
less than about or about 23 minutes, less than about or about 22
minutes, less than about or about 21 minutes, less than about or
about 20 minutes, less than about or about 19 minutes, less than
about or about 18 minutes, less than about or about 17 minutes,
less than about or about 16 minutes, less than about or about 15
minutes, less than about or about 14 minutes, less than about or
about 13 minutes, less than about or about 12 minutes, less than
about or about 11 minutes, or less than about or about 10
minutes.
[0062] As used herein, the term "hematopoietic compartment" refers
to the cell compartment in a subject that contains all blood cell
lineages, including without limitation, the myeloid lineage, which
includes, without limitation, monocytes, macrophages, neutrophils,
basophils, eosinophils, erythrocytes, megakaryocytes, platelets,
and dendritic cells; and the lymphoid lineage, which includes,
without limitation, T-cells, B-cells, NKT-cells, and NK cells. The
"hematopoietic compartment" can contain all immature, mature,
undifferentiated, and differentiated white blood cell populations
and sub-populations, including tissue-specific and specialized
varieties.
[0063] As used herein, the term "hematopoietic compartment cell
formation" in a subject refers to the production and/or expansion
of one or more cells of any blood cell lineages of the
hematopoietic compartment in the hematopoietic compartment from
hematopoietic stem cell (HSC) differentiation, HSC proliferation,
and/or HSC survival. "Hematopoietic compartment cell formation" may
be the result of HSC engraftment by exogenous HSCs, such as
hematopoietic compartment reconstitution in an HSC transplant
recipient. Alternatively, hematopoietic compartment cell formation"
may be the result of endogenous HSC differentiation, endogenous HSC
proliferation, and/or endogenous HSC survival, such as from
hematopoietic compartment autoreconstitution in a subject. In some
embodiments, "hematopoietic compartment cell formation" includes,
without limitation, one or more of myeloid lineage formation,
myeloid lineage progenitor cell formation, monocyte cell formation,
macrophage cell formations, neutrophil cell formation, basophil
cell formation, eosinophil cell formation, erythrocyte cell
formation, megakaryocyte cell formation, platelet cell formation,
dendritic cell formation, lymphoid lineage formation, lymphoid
lineage progenitor cell formation, T-cell formation, B-cell
formation, NKT-cell formation, and NK cell formation.
[0064] As used herein, "enhancing hematopoietic compartment cell
formation" in a subject refers to one or more of i) increasing the
rate of hematopoietic compartment cell formation (e.g.,
accelerating hematopoietic compartment reconstitution with
exogenous HSCs or accelerating autoreconstitution with endogenous
HSCs) by at least about or about 5% to at least about or about
500%, as compared to the rate of hematopoietic compartment cell
formation in a subject that is administered exogenous HSCs that
have not been treated with a MYC-composition, a Bcl-2 composition,
or both, or as compared to the rate of hematopoietic compartment
cell formation from HSCs in a subject that is not administered a
MYC-composition; ii) increasing the amount of hematopoietic
compartment cells that are formed in the hematopoietic compartment
of the subject from either exogenous or endogenous HSCs by at least
about or about 5% to at least about or about 500%, as compared to
the amount of hematopoietic compartment cells that are formed in
the hematopoietic compartment in a subject that is administered
exogenous HSCs that have not been treated with a MYC-composition, a
Bcl-2 composition, or both, or as compared to the amount of
hematopoietic compartment cells that are formed in the
hematopoietic compartment in a subject that is not administered a
MYC-composition, a Bcl-2 composition, or both; or iii) reducing
loss of hematopoietic compartment cells in the subject by at least
about or about 5% to at least about or about 500%, as compared to
the amount of hematopoietic compartment cells that are lost in a
subject that is administered exogenous HSCs that have not been
treated with a MYC-composition, a Bcl-2 composition, or both, or as
compared to the amount of hematopoietic compartment cells that are
lost in a subject that is not administered a MYC-composition, a
Bcl-2 composition or both. Moreover, "enhancing hematopoietic
compartment cell formation" in a subject includes, without
limitation, enhancing hematopoietic compartment reconstitution from
exogenous HSCs (e.g., HSC engraftment), and enhancing hematopoietic
compartment autoreconstitution from endogenous HSCs. As used
herein, "loss of hematopoietic compartment cells" in a subject
refers to a reduction in the amount of hematopoietic compartment
cells due to cell necrosis, apoptosis, and the like.
[0065] Similarly, hematopoietic compartment cell formation in a
subject is enhanced when: i) the rate of hematopoietic compartment
cell formation is increased (e.g., hematopoietic compartment
reconstitution with exogenous HSCs is accelerated or
autoreconstitution with endogenous HSCs is accelerated), for
example, by at least about or about 5% to at least about or about
500%, as compared to the rate of hematopoietic compartment cell
formation in a subject that is administered exogenous HSCs that
have not been treated with a MYC-composition, a Bcl-2-composition,
or both, or as compared to the rate of hematopoietic compartment
cell formation from HSCs in a subject that is not administered a
MYC-composition; ii) the amount of hematopoietic compartment cells
that are formed in the hematopoietic compartment of the subject is
increased, for example, by at least about or about 5% to at least
about or about 500%, as compared to the amount of hematopoietic
compartment cells that are formed in the hematopoietic compartment
in a subject that is administered exogenous HSCs that have not been
treated with a MYC-composition, a Bcl-2-composition, or both, or as
compared to the amount of hematopoietic compartment cells that are
formed in the hematopoietic compartment in a subject that is not
administered a MYC-composition; and/or iii) loss of hematopoietic
compartment cells in the subject is reduced, for example, by at
least about or about 5% to at least about or about 500%, as
compared to the amount of hematopoietic compartment cells that are
lost in a subject that is administered exogenous HSCs that have not
been treated with a MYC-composition, a Bcl-2-composition, or both,
or as compared to the amount of hematopoietic compartment cells
that are lost in a subject that is not administered a
MYC-composition.
[0066] Any method known in the art and disclosed herein for
measuring the rate of hematopoietic compartment cell formation
(e.g., hematopoietic compartment reconstitution or
autoreconstitution) from HSCs, for measuring the amount of
hematopoietic compartment cells that are formed in the
hematopoietic compartment of a subject, and/or for measuring loss
of hematopoietic compartment cells in a subject may be used. In one
non-limiting example, fluorescent-tagged antibodies specific for
blood cell lineage marker and fluorescence-activated flow cytometry
(FACS) analysis is utilized.
[0067] In certain embodiments, the rate of hematopoietic
compartment cell formation is considered to be increased when the
rate of hematopoietic compartment cell formation is increased, for
example, by at least about or about 5%, at least about or about
10%, at least about or about 15%, at least about or about 20%, at
least about or about 25%, at least about or about 30%, at least
about or about 31%, at least about or about 32%, at least about or
about 33%, at least about or about 34%, at least about or about
35%, at least about or about 40%, at least about or about 45%, at
least about or about 50%, at least about or about 55%, at least
about or about 60%, at least about or about 65%, at least about or
about 66%, at least about or about 67%, at least about or about
68%, at least about or about 69%, at least about or about 70%, at
least about or about 75%, at least about or about 80%, at least
about or about 90%, at least about or about 95%, at least about or
about 100%, at least about or about 150%, at least about or about
200%, at least about or about 250%, at least about or about 300%,
at least about or about 400%, at least about or about 500%, or a
higher percentage, as compared to the rate of hematopoietic
compartment cell formation in a subject that is administered
exogenous HSCs that have not been treated with a MYC-composition, a
Bcl-2-composition, or both, or as compared to the rate of
hematopoietic compartment cell formation from HSCs in a subject
that is not administered a MYC-composition.
[0068] In certain embodiments, the amount of hematopoietic
compartment cells that are formed in the hematopoietic compartment
of a subject is considered to be increased when the amount of
hematopoietic compartment cells formed is increased, for example,
by at least about or about 5%, at least about or about 10%, at
least about or about 15%, at least about or about 20%, at least
about or about 25%, at least about or about 30%, at least about or
about 31%, at least about or about 32%, at least about or about
33%, at least about or about 34%, at least about or about 35%, at
least about or about 40%, at least about or about 45%, at least
about or about 50%, at least about or about 55%, at least about or
about 60%, at least about or about 65%, at least about or about
66%, at least about or about 67%, at least about or about 68%, at
least about or about 69%, at least about or about 70%, at least
about or about 75%, at least about or about 80%, at least about or
about 90%, at least about or about 95%, at least about or about
100%, at least about or about 150%, at least about or about 200%,
at least about or about 250%, at least about or about 300%, at
least about or about 400%, at least about or about 500%, or a
higher percentage, as compared to the amount of hematopoietic
compartment cells that are formed in the hematopoietic compartment
in a subject that is administered exogenous HSCs that have not been
treated with a MYC-composition, a Bcl-2-composition, or both, or as
compared to the amount of hematopoietic compartment cells that are
formed in the hematopoietic compartment in a subject that is not
administered a MYC-composition.
[0069] In certain embodiments, loss of hematopoietic compartment
cells in a subject is considered to be decreased when the loss of
cells is reduced, for example, by at least about or about 5%, at
least about or about 10%, at least about or about 15%, at least
about or about 20%, at least about or about 25%, at least about or
about 30%, at least about or about 31%, at least about or about
32%, at least about or about 33%, at least about or about 34%, at
least about or about 35%, at least about or about 40%, at least
about or about 45%, at least about or about 50%, at least about or
about 55%, at least about or about 60%, at least about or about
65%, at least about or about 66%, at least about or about 67%, at
least about or about 68%, at least about or about 69%, at least
about or about 70%, at least about or about 75%, at least about or
about 80%, at least about or about 90%, at least about or about
95%, at least about or about 100%, at least about or about 150%, at
least about or about 200%, at least about or about 250%, at least
about or about 300%, at least about or about 400%, at least about
or about 500%, or a higher percentage, as compared to the amount of
hematopoietic compartment cells that are lost in a subject that is
administered exogenous HSCs that have not been treated with a
MYC-composition, a Bcl-2-composition, or both, or as compared to
the amount of hematopoietic compartment cells that are lost in a
subject that is not administered a MYC-composition.
MYC-Compositions
[0070] Certain aspects of the present disclosure relate to treating
a population of hematopoietic stem cells (HSCs) with a composition
containing a MYC-composition to enhance hematopoietic compartment
reconstitution. HSCs of the present disclosure may be treated with
the MYC-composition alone, or in combination with a
Bcl-2-composition of the present disclosure. Any method of treating
cells with a composition, such as fusion protein, known in the art
and disclosed herein may be used. For example, a population of
hematopoietic stem cells may be cultured in the presence of the
MYC-composition. Other aspect of the present disclosure relate to
administering to a subject in need thereof, a composition
containing a MYC-composition to enhance hematopoietic compartment
formation in the subject.
[0071] As used herein, a "MYC-composition" refers to a MYC
polypeptide; a variant or mutant of a MYC polypeptide, a modified
MYC polypeptide, a homologue of a MYC polypeptide; an analogue of a
MYC polypeptide; a biologically active fragment of a MYC
polypeptide; a downstream target of a MYC polypeptide, a homologue
thereof, an analogue thereof, or a biologically active fragment
thereof, and a fusion protein containing a MYC polypeptide, a
homologue thereof, an analogue thereof, and a biologically active
fragment thereof. A MYC-composition of the present disclosure
includes any MYC polypeptide, variant thereof, mutant thereof,
homologue thereof, analogue thereof, or biologically active
fragment thereof known in the art (e.g., US Patent Application
Publication Nos. US 2007/0116691, US 2009/0291094, US 2010/0047217,
US 2010/0055129, and US 2010/0279351).
[0072] In certain preferred embodiments, MYC-compositions of the
present disclosure are fusion proteins that contain a MYC
polypeptide, variant thereof, mutant thereof, homologue thereof,
analogue thereof, or biologically active fragment thereof that has
been coupled (e.g., fused) to a protein transduction domain
(PTD).
[0073] MYC Polypeptides
[0074] In some embodiments, a MYC-composition of the present
disclosure is a MYC polypeptide. A MYC polypeptide of the present
disclosure includes, without limitation, any polypeptide having one
or more activities of a full-length MYC protein.
[0075] As used herein, "MYC" and "MYC protein" are used
interchangeably and refer to a protein that is a member of the MYC
family of bHLH (basic helix-loop-helix) transcription factors. MYC
proteins of the present disclosure are transcription factors that
regulate expression of MYC responsive genes, and as such are
required to enter the nucleus of a cell to function as
transcription factors. MYC activity can activate expression of
certain MYC responsive genes, while repressing expression of other
MYC responsive genes. MYC activity can regulate various cellular
functions including, without limitation, cell proliferation, cell
growth, and apoptosis.
[0076] MYC-compositions of the present disclosure allow for an
increase in MYC activity in a subject in need of hematopoietic
compartment cell formation by the exogenous addition of MYC,
without the need for overexpressing endogenous MYC or recombinantly
expressing MYC via genetic manipulation.
[0077] MYC polypeptides of the present disclosure include, without
limitation, full-length MYC proteins, fragments of MYC proteins
that retain at least one activity of a full-length MYC protein,
homologs thereof that retain at least one activity of a full-length
MYC protein, and analogs thereof that retain at least one activity
of a full-length MYC protein. MYC polypeptides of the present
disclosure may be produced by any suitable method known in the art.
For example, a MYC polypeptide may be purified from a native
source, may be recombinantly expressed, or may be chemically
synthesized.
[0078] MYC Proteins
[0079] Examples of full-length MYC proteins suitable for use in any
of the methods of the present disclosure include, without
limitation, c-Myc, N-Myc, L-Myc, and S-Myc.
[0080] In certain preferred embodiments, the MYC polypeptide is a
full-length c-Myc polypeptide. The c-Myc polypeptide may have one
or more of the following features: the polypeptide may be a polymer
of 439 amino acids, the polypeptide may have a molecular weight of
48,804 kDa, the polypeptide may contain a basic Helix-Loop-Helix
Leucine Zip-per (bHLH/LZ) domain, or the polypeptide may bind to a
sequence containing CACGTG (i.e., an E-box sequence). Preferably,
the c-Myc polypeptide is the human c-Myc polypeptide having NCBI
Accession Number NP_002458.2. Moreover, a c-Myc polypeptide of the
present disclosure may be a c-Myc polypeptide that has not
undergone any post-translational modifications. Alternatively, a
c-Myc polypeptide of the present disclosure may be a c-Myc
polypeptide that has undergone post-translational
modifications.
[0081] In some embodiments, the MYC polypeptide is a fusion protein
containing a protein transduction domain (PTD). In certain
embodiments, the MYC polypeptide is a fusion protein containing a
TAT protein, or fragment thereof, of the present disclosure. In
some embodiments, the MYC polypeptide is a TAT-MYC fusion protein
with the following amino acid sequence (SEQ ID NO: 1):
TABLE-US-00001 MRKKRRQRRRMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSE
LQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDG
GGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSG
FSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAAS
FCIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSP
EPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSA
GGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVL
RQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIP
ELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKL
EQLRKGELNSKLEGKPIPNPLLGLDSTRTGHHHHHH
[0082] In the TAT-MYC polypeptide of SEQ ID NO: 1, amino acids 2-10
correspond to the HIV TAT protein transduction domain, amino acids
11-454 correspond to the amino acid sequence of c-Myc, amino acids
455-468 correspond to a 14 amino acid V5 epitope, and amino acids
472-477 correspond to a 6 Histidine tag (SEQ ID NO: 6).
[0083] Biologically Active MYC Fragments
[0084] In other embodiments, a MYC-composition of the present
disclosure is a biologically active fragment of a full-length MYC
protein that retains at least one activity of a full-length MYC
protein. The MYC polypeptide may be a fragment of c-Myc, N-Myc,
L-Myc, or S-Myc.
[0085] A MYC fragment of the present disclosure may contain at
least 10, at least 15, at least 20, at least 25, at least 30, at
least 35, at least 40, at least 45, at least 50, at least 55, at
least 60, at least 65, at least 70, at least 75, at least 80, at
least 85, at least 90, at least 95, at least 100, at least 150, at
least 200, at least 250, at least 300, at least 350, at least 400,
or more consecutive amino acid residues of the amino acid sequence
of a MYC protein.
[0086] MYC Homologues
[0087] In other embodiments, a MYC-composition of the present
disclosure is a homologue of a MYC protein, or a homologue of a
fragment thereof that retains at least one activity of a
full-length MYC protein.
[0088] For example, a MYC polypeptide of the present disclosure may
include an amino acid sequence that is at least 40% to 100%
identical, e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or
any other percent from about 40% to about 100% identical to a MYC
protein or fragments thereof.
[0089] In certain embodiments, the MYC polypeptide is a homologue
of c-Myc, N-Myc, L-Myc, S-Myc, or fragments thereof.
[0090] MYC polypeptides of the present disclosure also include
functional homologs or analogs of the human c-Myc polypeptide
having NCBI Accession Number NP_002458.2, or fragment thereof. In
certain embodiments, the c-Myc homolog or analog contains an amino
acid sequence that is at least 40% to 100% identical, e.g., at
least 40%, 45%. 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%,
88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other percent
from about 40% to about 100% identical to the c-Myc polypeptide
sequence of NCBI Accession Number NP_002458.2 or a fragment
thereof.
[0091] In other embodiments, the c-Myc homolog or analog contains a
polypeptide sequence of at least 10 amino acids, at least 20 amino
acids, at least 30 amino acids, at least 40 amino acids, at least
50 amino acids, at least 60 amino acids, at least 70 amino acids,
at least 80 amino acids, at least 90 amino acids, at least 100
amino acids, at least 150 amino acids, at least 200 amino acids, at
least 250 amino acids, at least 300 amino acids, at least 350 amino
acids, at least 400 amino acids, or more in length that is at least
50% to 100% identical, e.g., at least 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or
any other percent from about 50% to about 100% identical to the
c-Myc polypeptide sequence of NCBI Accession Number NP_002458.2 or
a fragment thereof.
[0092] As used herein, a "homologue" refers to a protein or
polypeptide having amino acid sequence similarity between a
reference sequence and at least a fragment of a second sequence.
Homologues may be identified by any method known in the art,
preferably, by using the BLAST tool to compare a reference sequence
to a single second sequence or fragment of a sequence or to a
database of sequences. As described below, BLAST will compare
sequences based upon percent identity and similarity.
[0093] The terms "identical" or percent "identity," in the context
of two or more sequences (e.g., amino acid sequences), refer to two
or more sequences or subsequences that are the same. Two sequences
are substantially identical if two sequences have a specified
percentage of amino acid residues or nucleotides that are the same
(i.e., 29% identity, optionally 30%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity over a specified
region, or, when not specified, over the entire sequence), when
compared and aligned for maximum correspondence over a comparison
window, or designated region as measured using one of the following
sequence comparison algorithms or by manual alignment and visual
inspection. Optionally, the identity exists over a region that is
at least about 10 amino acids in length, or more preferably over a
region that is 20, 50, 200, or more amino acids in length.
[0094] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters. When comparing two sequences for identity, it
is not necessary that the sequences be contiguous, but any gap
would carry with it a penalty that would reduce the overall percent
identity. For blastp, the default parameters are Gap opening
penalty=11 and Gap extension penalty=1. For blastn, the default
parameters are Gap opening penalty=5 and Gap extension
penalty=2.
[0095] As used herein, a "comparison window" includes reference to
a segment of any one of the number of contiguous positions
including, but not limited to from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith and
Waterman (1981), by the homology alignment algorithm of Needleman
and Wunsch (1970) J Mol Biol 48(3):443-453, by the search for
similarity method of Pearson and Lipman (1988) Proc Natl Acad Sci
USA 85(8):2444-2448, by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by manual alignment and visual inspection
[see, e.g., Brent et al., (2003) Current Protocols in Molecular
Biology, John Wiley & Sons, Inc. (Ringbou Ed)].
[0096] Two examples of algorithms that are suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al. (1997)
Nucleic Acids Res 25(17):3389-3402 and Altschul et al. (1990) J.
Mol Biol 215(3)-403-410, respectively. Software for performing
BLAST analyses is publicly available through the National Center
for Biotechnology Information. This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. For amino acid
sequences, the BLASTP program uses as defaults a wordlength of 3,
and expectation (E) of 10, and the BLOSUM62 scoring matrix [see
Henikoff and Henikoff, (1992) Proc Natl Acad Sci USA
89(22):10915-10919] alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands. For nucleotide
sequences, the BLASTN program uses as defaults a wordlength (W) of
11, an expectation (E) or 10, M=5, N=-4, and a comparison of both
strands.
[0097] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin and
Altschul, (1993) Proc Natl Acad Sci USA 90(12):5873-5877). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0098] Other than percentage of sequence identity noted above,
another indication that two polypeptides are substantially
identical is that the first polypeptide is immunologically
cross-reactive with antibodies raised against the second
polypeptide. Thus, a polypeptide is typically substantially
identical to a second polypeptide, for example, where the two
peptides differ only by conservative substitutions.
[0099] As disclosed herein, suitable MYC polypeptides also include
conservatively modified variants of MYC polypeptides of the present
disclosure. "Conservatively modified variants" as used herein
include individual substitutions, deletions, or additions to an
encoded amino acid sequence which result in the substitution of an
amino acid with a chemically similar amino acid. Conservative
substitution tables providing functionally similar amino acids are
well known in the art. The following eight groups contain amino
acids that are conservative substitutions for one another: 1)
Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)
Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)
Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),
Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,
Creighton, Proteins (1984)).
[0100] MYC Analogues,
[0101] In other embodiments, a MYC-composition of the present
disclosure is an analogue of a MYC protein, or an analogue of a
fragment thereof that retains at least one activity of a
full-length MYC protein. In certain embodiments, the MYC
polypeptide is an analogue of c-Myc, N-Myc, L-Myc, S-Myc, or
fragments thereof.
[0102] As used herein, an "analogue" refers to a protein that
structurally or functionally resembles a protein of interest, such
as a MYC polypeptide. Compared to the starting protein, an analogue
may exhibit the same, similar, or improved activity and/or
function. Methods of screening for analogues and/or synthesizing
analogues are well known in the art.
[0103] Suitable MYC analogues of the present disclosure include,
without limitation, HIF-1a and ICN-1.
[0104] Proteins Downstream of MYC
[0105] In other embodiments, a MYC-composition of the present
disclosure is a protein that is downstream of MYC in a MYC pathway.
Any protein downstream known in the art is suitable for use with
the methods of the present disclosure. Examples of suitable
proteins that are downstream of MYC include, without limitation,
AKT and AKT-related proteins, such as PDK-1, mTORC2, PI3K-delta.
The protein downstream of MYC t may further include a protein
transduction domain (PTD). Accordingly, in certain embodiments, the
protein downstream of MYC is an AKT-PTD fusion protein, a PTD-PDK-1
fusion protein, a PTD-mTORC2 fusion protein, or a PTD-PI3K-delta
fusion protein.
[0106] In other embodiments, hematopoietic compartment
reconstitution is enhanced in a subject in need thereof by
administering HSCs that have been pre-treated with an inhibitor
(e.g., genetic, chemical, small molecule, etc.) that inhibits a
protein that antagonizes HSC survival and/or proliferation and that
is downstream of MYC. Similarly, hematopoietic compartment
formation is enhanced in a subject in need thereof by administering
a therapeutically effective amount of a composition containing
inhibitor (e.g., genetic, chemical, small molecule, etc.) that
inhibits a protein that antagonizes HSC survival and/or
proliferation and that is downstream of MYC. Methods of
administering therapeutically effective amounts of compositions,
such as those containing inhibitors, and determining therapeutic
amounts are well known in the art and described herein. Examples of
proteins that antagonize HSC survival and/or proliferation and that
are downstream of MYC include, without limitation, of pTEN, PP2A,
PHLPP, CTMP. Any method known in the art for inhibiting protein
and/or gene expression, activity, and/or function may be used,
including without limitation the methods disclosed herein.
Non-limiting examples include genetic inhibitors, small molecule
inhibitors, RNA interference, and antibodies.
[0107] Activities of Full-Length MYC Proteins
[0108] In other embodiments, a MYC-composition of the present
disclosure contains a full-length MYC polypeptide having at least
one MYC activity, a fragment of a MYC protein that retains at least
one activity of a full-length MYC protein, a homologue of a MYC
protein that retains at least one activity of a full-length MYC
protein, or an analogue of a MYC protein that retains at least one
activity of a full-length MYC protein.
[0109] Full-length MYC proteins of the present disclosure have
numerous activities. Examples of such activities include, without
limitation, transcription factor activity, protein binding
activity, nucleic acid binding activity, cell proliferation
regulation activity, cell growth regulation activity, apoptosis
regulation activity, morphogenesis regulation activity, development
regulation activity, and enhanced hematopoietic compartment
reconstitution activity.
[0110] In some embodiments a MYC-composition of the present
disclosure has a MYC activity that enhances hematopoietic
compartment reconstitution in an HSC transplant recipient,
including, without limitation, a bone marrow transplant recipient.
Without wishing to be bound by theory, it is believed that MYC
activity enhances productive homing of the transplanted HSCs to
bone marrow niches in the recipient. The recovery time of mature
hematopoietic lineages following HSC transplantation is largely
dependent of the ability of the administered HSCs to home to bone
marrow niches. Once the HSCs arrive at the bone marrow niches, they
need to establish a molecular crosstalk with the niche-resident
cells. This crosstalk is thought to regulate the nature and levels
of cell-intrinsic signals within the HSCs that regulate their
survival, proliferation, self-renewal, and differentiation. While
the full extent of the surface molecules that regulate the homing
and polarized cell division process between bone marrow niches and
HSCs is not yet fully understood, it is clear that several adhesion
molecules, such as P-selectin and E-selectin, are involved in this
process. Without wishing to be bound by theory, it is believed that
MYC is a key regulator of expression of P-selectin and E-selectin,
in addition to PSGL1, VLA4, VLA5, LFA1, and CD26 in the homing and
maintenance of HSCs at bone marrow niches. It is also believed that
MYC is a regulator of survival and differentiation signals within
the HSCs. Further, it is believed that MYC regulates the expression
and/or function of several additional pathways, such as the Wnt
pathway, that are involved in the maintenance of HSC pluripotency
and self-renewal. This molecular interplay enables the HSCs to
resume a near-quiescent state that supports their asymmetric cell
division, allowing for the generation of short-term HSCs that give
rise to mature hematopoietic lineages, and for a long-term HSC
compartment that enables the successful reconstitution of the
hematopoietic lineages for the lifetime of the HSC transplant
recipient. Thus, administering a MYC-composition of the present
disclosure to an HSC transplant recipient results in an at least a
50% increase in HSC productive homing to bone marrow niches in the
recipient.
[0111] In further embodiments, a MYC-composition of the present
disclosure contains a MYC polypeptide whose activity enhances
hematopoietic compartment autoreconstitution in a subject in need
of hematopoietic compartment autoreconstitution. Without wishing to
be bound by theory, it is believed that MYC activity enhances the
ability of endogenous HSCs to self-renew and to differentiate into
all hematopoietic compartment lineages, thus enhancing
hematopoietic compartment autoreconstitution. Without wishing to be
bound by theory, it is also believed that MYC activity enhances HSC
homing to their bone marrow niches.
[0112] Advantageously, administering MYC in the form of a
MYC-composition results in transient MYC activity in a subject.
This transient MYC activity avoids the potentially negative effects
of prolonged MYC activity, such as oncogenicity.
[0113] Additionally, MYC-compositions of the present disclosure can
increase the intracellular levels of MYC in both endogenous and
exogenously transplanted HSCs and committed lineage precursors.
Thus, in certain embodiments, administering a MYC-composition of
the present disclosure to a subject in need of hematopoietic
compartment autoreconstitution results in an expansion of
endogenous HSCs. In other embodiments, administering a
MYC-composition of the present disclosure to an HSC transplant
recipient, such as a bone marrow transplant recipient, results in
an expansion of the transplanted HSCs.
[0114] As disclosed herein, a therapeutically effective amount a
MYC-composition of the present disclosure is at least about or
about 0.5.mu./ml to at least about or about 100.mu./ml, for example
at least about or about 0.5.mu./ml, at least about or about
0.6.mu./ml, at least about or about 0.7.mu./ml, at least about or
about 0.8.mu./ml, at least about or about 0.9.mu./ml, at least
about or about 1.mu./ml, at least about or about 2.mu./ml, at least
about or about 3.mu./ml, at least about or about 4.mu./ml, at least
about or about 5.mu./ml, at least about or about 6.mu./ml, at least
7 about or about p/ml, at least about or about 8.mu./ml, at least
about or about 9.mu./ml, at least about or about 10.mu./ml, at
least about or about 15.mu./ml, at least about or about 20.mu./ml,
at least about or about 25.mu./ml, at least about or about
30.mu./ml, at least about or about 35.mu./ml, at least about or
about 40.mu./ml, at least about or about 45.mu./ml, at least about
or about 50.mu./ml, at least about or about 55.mu./ml, at least
about or about 60.mu./ml, at least about or about 65.mu./ml, at
least about or about 70.mu./ml, at least about or about 75.mu./ml,
at least about or about 80.mu./ml, at least about or about
85.mu./ml, at least about or about 90.mu./ml, at least about or
about 95.mu./ml, or at least about or about 100.mu./ml of the
MYC-composition.
Bcl-2-Compositions
[0115] Certain aspects of the present disclosure relate to treating
a population of hematopoietic stem cells (HSCs) with a composition
containing a Bcl-2-composition to enhance hematopoietic compartment
reconstitution. HSCs of the present disclosure may be treated with
the Bcl-2-composition alone, or in combination with a
MYC-composition of the present disclosure. Any method of treating
cells with a composition, such as fusion protein, known in the art
and disclosed herein may be used. For example, a population of
hematopoietic stem cells may be cultured in the presence of the
Bcl-2-composition. In some embodiments, a population of
hematopoietic stem cells may be treated with a combination of a MYC
composition of the present disclosure along with a
Bcl-2-composition of the present disclosure. Other aspect of the
present disclosure relate to administering to a subject in need
thereof, a composition containing a Bcl-2-composition to enhance
hematopoietic compartment formation in the subject.
[0116] In some embodiments, a composition containing a
Bcl-2-composition of the present disclosure may be administered to
a subject in need thereof, to enhance hematopoietic compartment
formation in the subject. Without wishing to be bound by theory, it
is believed that the Bcl-2-composition may keep more exogenous
and/or endogenous hematopoietic stem cells alive (i.e., increase
cell survival) long enough to enhance hematopoietic cell
compartment formation (e.g., hematopoietic compartment
reconstitution and/or engraftment by exogenous HSCs or
autoreconstitution by endogenous HSCs). Exemplary methods of
determining hematopoietic cell compartment formation are disclosed
herein and known in the art. The Bcl-2-composition may be
administered in addition to or instead of a MYC-composition of the
present disclosure.
[0117] As used herein, a "Bcl-2-composition" refers to a Bcl-2
polypeptide; a variant or mutant of a Bcl-2 polypeptide, a modified
Bcl-2 polypeptide, a homologue of a Bcl-2 polypeptide; an analogue
of a Bcl-2 polypeptide; a biologically active fragment of a Bcl-2
polypeptide; a downstream target of a Bcl-2 polypeptide, a
homologue thereof, an analogue thereof, or a biologically active
fragment thereof; and a fusion protein containing a Bcl-2
polypeptide, a homologue thereof, an analogue thereof, and a
biologically active fragment thereof. A Bcl-2-composition of the
present disclosure includes any Bcl-2 polypeptide, variant thereof,
mutant thereof, homologue thereof, analogue thereof, or
biologically active fragment thereof known in the art (e.g., US
Patent Application Publication Nos. US 2007/0116691, US
2010/0047217, and US 2010/0279351).
[0118] In certain preferred embodiments, Bcl-2-compositions of the
present disclosure are fusion proteins that contain a Bcl-2
polypeptide, variant thereof, mutant thereof, homologue thereof,
analogue thereof, or biologically active fragment thereof that has
been coupled (e.g., fused) to a protein transduction domain
(PTD).
[0119] Bcl-2 Polypeptides
[0120] In some embodiments, a Bcl-2-composition of the present
disclosure is a Bcl-2 polypeptide. A Bcl-2 polypeptide of the
present disclosure includes, without limitation, any polypeptide
having the activity of a Bcl-2 protein.
[0121] As used herein, "Bcl-2," "Bcl-2 polypeptide," and "Bcl-2
protein" are used interchangeably and refer to a protein that is a
member of the Bcl-2 protein family that has one or more and/or all
Bcl-2 homology (BH) domains, such as but not limited to, BH1, BH2,
BH3, and BH4. Members of the bcl-2 protein family typically form
heterodimer or homodimers, and function as regulators of apoptosis.
In certain preferred embodiments, Bcl-2 polypeptides of the present
disclosure have anti-apoptotic activity.
[0122] Bcl-2-compositions of the present disclosure may allow for
an increase in Bcl-2 activity in a subject in need of hematopoietic
compartment cell formation by the exogenous addition of Bcl-2,
without the need for overexpressing endogenous Bcl-2 or
recombinantly expressing Bcl-2 via genetic manipulation.
[0123] Bcl-2 polypeptides of the present disclosure include,
without limitation, full length Bcl-2 proteins, fragments that
retain the activity of a full-length Bcl-2 protein, homologues
thereof, and analogues thereof. In some embodiments, Bcl-2
fragments that retain the activity of a full-length Bcl-2 protein
include a truncated form of Bcl-2 that has been deleted for the
unstructured loop domain (Anderson, M., et al. (1999). Refolding,
purification and characterization of a loop deletion mutant of
human Bcl-2 from bacterial inclusion bodies. Prot Expr. Purif. 15,
162-70). Bcl-2 polypeptides of the present disclosure may be
produced by any suitable method known in the art. For example, a
Bcl-2 polypeptide may be purified from a native source, may be
recombinantly expressed, or may be chemically synthesized.
[0124] Bcl-2 Proteins
[0125] Examples of full length Bcl-2 proteins suitable for use in
any of the methods of the present disclosure include, without
limitation, Bcl-2, Bcl-x, Bcl-XL, Mcl-1, CED-9, Bcl-2 related
protein A1, Bfl-1, and Bcl-w.
[0126] In certain preferred embodiments, the Bcl-2 polypeptide is a
full-length human Bcl-2 polypeptide that has been deleted for the
unstructured loop domain. The human Bcl-2 polypeptide may have one
or more of the following features: the polypeptide may be a polymer
of 239 amino acids, the polypeptide may have a molecular weight of
approximately 26.3 kDa, or the polypeptide may contain at least one
Bcl-2 homology (BH) domain, such as BH1, BH2, BH3, and BH4.
Preferably, the human Bcl-2 polypeptide is the Bcl-2 polypeptide
having NCBI Accession Number NP_000624.2. Moreover, a Bcl-2
polypeptide of the present disclosure may be a Bcl-2 polypeptide
that has not undergone any post-translational modifications.
Alternatively, a Bcl-2 polypeptide of the present disclosure may be
a Bcl-2 polypeptide that has undergone post-translational
modifications.
[0127] In some embodiments, the Bcl-2 polypeptide is a fusion
protein containing a protein transduction domain (PTD). In certain
embodiments, the Bcl-2 polypeptide is a fusion protein containing a
TAT protein, or fragment thereof, of the present disclosure. In
some embodiments, the Bcl-2 polypeptide is a TAT-Bcl-2.DELTA.
fusion protein, where the Bcl-2 polypeptide has a deletion of the
unstructured loop domain. In certain embodiments, the
TAT-Bcl-2.DELTA. fusion protein has the following amino acid
sequence (SEQ ID NO: 3):
TABLE-US-00002 MRKKRRQRRRMAHAGRSGYDNREIVMKYIHYKLSQRATSGISIEAAGPA
LSPVPPVVHLTLRQAGDDFSRRYRRDFAEMSSQLHLTPFTARGCFATVV
EELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIALWMTEYLNR
HLHTWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKTLLSLALVGACITL
GAYLSHKKGELNSKLEGKPIPNPLLGLDSTRTGHHHHHH
[0128] In the TAT-Bcl-2.DELTA. polypeptide of SEQ ID NO: 3, amino
acids 2-10 correspond to the HIV TAT protein transduction domain,
amino acids 11-212 correspond to the amino acid sequence of Bcl-2A,
amino acids 4213-226 correspond to a 14 amino acid V5 epitope, and
amino acids 230-235 correspond to a 6 Histidine tag (SEQ ID NO:
6).
[0129] Biologically Active Bcl-2 Fragments
[0130] In other embodiments, a Bcl-2-composition of the present
disclosure is a biologically active fragment of a full-length Bcl-2
protein that retains at least one activity of a full-length Bcl-2
protein. The Bcl-2 polypeptide may be a fragment of Bcl-2, Bcl-x,
Bcl-XL, Mcl-1, CED-9, Bcl-2 related protein A1, Bfl-1, or
Bcl-w.
[0131] A Bcl-2 fragment of the present disclosure may contain at
least 10, at least 15, at least 20, at least 25, at least 30, at
least 35, at least 40, at least 45, at least 50, at least 55, at
least 60, at least 65, at least 70, at least 75, at least 80, at
least 85, at least 90, at least 95, at least 100, at least 110, at
least 120, at least 130, at least 140, at least 150, at least 160,
at least 170, at least 180, at least 190, at least 200, at least
210, at least 220, at least 230, or more consecutive amino acid
residues of the amino acid sequence of a Bcl-2 protein.
[0132] Bcl-2 Homologues and Analogues
[0133] In other embodiments, a Bcl-2-composition of the present
disclosure is a homologue or analogue of a Bcl-2 protein or
fragment thereof. For example, a Bcl-2 polypeptide of the present
disclosure may include an amino acid sequence that is at least 40%
to 100% identical, e.g., at least 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%,
97%, 98%, or any other percent from about 40% to about 100%
identical to a Bcl-2 protein or fragments thereof. In certain
embodiments, the Bcl-2 polypeptide is a homologue or analogue of
Bcl-2, Bcl-x, Bcl-XL, Mcl-1, CED-9, Bcl-2 related protein A1,
Bfl-1, Bcl-w, or fragments thereof.
[0134] Bcl-2 polypeptides of the present disclosure also include
functional homologues or analogues of the human Bcl-2 polypeptide
having NCBI Accession Number NP_00624.2, or a fragment thereof. In
certain embodiments, the Bcl-2 homologue or analogue contains an
amino acid sequence that is at least 40% to 100% identical, e.g.,
at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%,
87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other
percent from about 40% to about 100% identical to the Bcl-2
polypeptide sequence of NCBI Accession Number NP_00624.2 or
fragment thereof.
[0135] In other embodiments, the Bcl-2 homologue or analogue
contains a polypeptide sequence of at least 10 amino acids, at
least 20 amino acids, at least 30 amino acids, at least 40 amino
acids, at least 50 amino acids, at least 60 amino acids, at least
70 amino acids, at least 80 amino acids, at least 90 amino acids,
at least 100 amino acids, at least 110 amino acids, at least 120
amino acids, at least 130 amino acids, at least 140 amino acids, at
least 150 amino acids, at least 160 amino acids, at least 170 amino
acids, at least 180 amino acids, at least 190 amino acids, at least
200 amino acids, at least 210 amino acids, at least 220 amino
acids, at least 230 amino acids, or more in length that is at least
50% to 100% identical, e.g., at least 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or
any other percent from about 50% to about 100% identical to the
Bcl-2 polypeptide sequence of NCBI Accession Number NP_00624.2 or
fragment thereof.
[0136] As disclosed herein, suitable Bcl-2 polypeptides also
include conservatively modified variants of Bcl-2 polypeptides of
the present disclosure.
[0137] Activities of Full-Length Bcl-2 Proteins
[0138] In other embodiments, a Bel-2-composition of the present
disclosure contains a full-length Bcl-2 polypeptide having at least
one Bcl-2 activity, a fragment of a Bcl-2 protein that retains at
least one activity of a full-length Bcl-2 protein, a homologue of a
Bcl-2 protein that retains at least one activity of a full-length
Bcl-2 protein, or an analogue of a Bcl-2 protein that retains at
least one activity of a full-length Bcl-2 protein.
[0139] Full-length Bcl-2 proteins of the present disclosure have
numerous activities. Examples of such activities include, without
limitation, apoptosis regulation activity, cell survival regulation
activity, protein binding activity, mitochondrial membrane
permeability regulation activity, caspase regulation activity,
voltage-dependent anion channel regulation activity, G2 checkpoint
regulation activity, outer mitochondrial membrane channel (VDAC)
regulation activity, mitochondrial membrane potential regulation
activity, protein channel activity, and cytochrome C regulation
activity.
[0140] In some embodiments a Bcl-2-composition of the present
disclosure has a Bcl-2 activity that either alone or in combination
with a MYC-composition enhances hematopoietic compartment
reconstitution in an HSC transplant recipient, including, without
limitation, a bone marrow transplant recipient, when the HSCs are
treated with the compositions prior to HSC transplantation.
[0141] As disclosed herein, a therapeutically effective amount a
Bcl-2-composition of the present disclosure is at least about or
about 0.5.mu./ml to at least about or about 100.mu./ml, for example
at least about or about 0.5.mu./ml, at least about or about
0.6.mu./ml, at least about or about 0.7.mu./ml, at least about or
about 0.8.mu./ml, at least about or about 0.9.mu./ml, at least
about or about 1.mu./ml, at least about or about 2.mu./ml, at least
about or about 3.mu./ml, at least about or about 4.mu./ml, at least
about or about 5.mu./ml, at least about or about 6.mu./ml, at least
7 about or about p/ml, at least about or about 8.mu./ml, at least
about or about 9.mu./ml, at least about or about 10.mu./ml, at
least about or about 15.mu./ml, at least about or about 20.mu./ml,
at least about or about 25.mu./ml, at least about or about
30.mu./ml, at least about or about 35.mu./ml, at least about or
about 40.mu./ml, at least about or about 45.mu./ml, at least about
or about 50.mu./ml, at least about or about 55.mu./ml, at least
about or about 60.mu./ml, at least about or about 65.mu./ml, at
least about or about 70.mu./ml, at least about or about 75.mu./ml,
at least about or about 80.mu./ml, at least about or about
85.mu./ml, at least about or about 90.mu./ml, at least about or
about 95.mu./ml, or at least about or about 100.mu./ml of the
Bcl-2-composition.
Protein Transduction Domains
[0142] Certain aspects of the present disclosure relate to fusion
proteins containing a protein transduction domain. In some
embodiments a MYC-composition of the present disclosure is a fusion
protein containing a protein transduction domain fused to a MYC
polypeptide. In other embodiments, a Bcl-2-composition of the
present disclosure is a fusion protein containing a protein
transduction domain fused to a Bcl-2 polypeptide.
[0143] As used herein, the terms "peptide transduction domain,"
"protein transduction domain," and "PTD" are used interchangeably
and refer to a peptide sequence or domain of a protein that
promotes penetration of protein into a mammalian cell and/or
compartment(s) within a mammalian cell. In one non-limiting
example, a PTD promotes penetration of a coupled peptide and/or
protein into the nucleus of a cell.
[0144] PTDs of the present disclosure may be isolated from a
PTD-containing protein by any method of isolating a protein domain
known in the art, such as standard molecular biology and
biochemical techniques. Alternatively, PTDs of the present
disclosure may be synthesized. Suitable PTDs of the present
disclosure may be about 8 to about 30 amino acid residues in
length, and enriched in basic amino acid residues, such as
argentine (Arg) and lysine (Lys).
[0145] Suitable PTDs of the present disclosure are not strongly
immunogenic, and as such do not induce a strong immune response
when administered to a subject. Moreover, PTDs of the present
disclosure also do not induce an immune response when administered
to an immunocompromised subject, such a subject who has received,
is receiving, or will receive a bone marrow or HSC transplant.
[0146] As disclosed herein, PTDs of the present disclosure are
coupled (e.g., fused, conjugated, cross-linked, etc.) to a peptide
and/or protein in order to facilitate the penetration of the
peptide and/or protein into a mammalian cell and/or compartment
within a mammalian cell. For example, in certain embodiments a PTD
of the present disclosure is coupled to a MYC protein.
[0147] Protein transduction domains suitable for use in any of the
methods of the present disclosure include any PTD known in the art
(e.g., U.S. Patent Application Publication Nos. US 2007/0116691 and
US 2010/0055129). For example, suitable PTDs may be obtained or
derived from proteins that include, without limitation, lentiviral
TAT (Trans-Activator of Transcription) proteins, lentiviral VPR
proteins, herpesviral VP22 proteins, and homeoproteins.
[0148] Examples of suitable PTDs obtained or derived from
lentiviral TAT proteins include, without limitation, the PTD from a
TAT protein of a TAT protein-containing virus, the PTD from a TAT
protein of a TAT protein-containing lentivirus, the PTD from the
HIV-1 TAT protein, the PTD from the HIV-2 TAT protein, the PTD from
the SIV TAT protein, the PTD from a primate lentivirus TAT protein,
the PTD from an ovine lentivirus TAT protein, the PTD from a bovine
lentivirus TAT protein, the PTD from an equine lentivirus TAT
protein, the PTD from a feline lentivirus TAT protein, a PTD from
the TAT protein of a subvariant of HIV, SIV, primate lentivirus,
ovine lentivirus, bovine lentivirus, equine lentivirus, or feline
lentivirus, and homologs thereof. In certain embodiments, the PTD
is amino acid residues 48-57 of the HIV TAT protein
(TAT.sub.[48-57]). In other embodiments, the PTD is amino acid
residues 57-48 of the HIV TAT protein (TAT.sub.[57-48]).
[0149] Examples of suitable PTDs that may obtained or derived from
lentiviral VPR proteins include, without limitation, the PTD from a
VPR protein of a VPR protein-containing virus, the PTD from a VPR
protein of a VPR protein-containing lentivirus, the PTD from the
HIV-1 VPR protein, the PTD from the HIV-2 VPR protein, the PTD from
the SIV VPR protein, the PTD from a primate lentivirus VPR protein,
the PTD from an ovine lentivirus VPR protein, the PTD from a bovine
lentivirus VPR protein, the PTD from an equine lentivirus VPR
protein, the PTD from a feline lentivirus VPR protein, a PTD from
the VPR protein of a subvariant of HIV, SIV, primate lentivirus,
ovine lentivirus, bovine lentivirus, equine lentivirus, or feline
lentivirus, and homologs thereof.
[0150] Examples of suitable PTDs that may obtained or derived from
herpesviral VP22 proteins include, without limitation, the PTD from
the human herpesvirus 1(HSV-1) VP22 protein, the PTD from the human
herpesvirus 2 (HSV-2) VP22 protein, the PTD from the BHV-1 VP22
protein, the PTD from the Psittacid herpesvirus 1VP22 protein, the
PTD from the Equine herpesvirus 1 VP22 protein, the PTD from the
Equine herpesvirus 4 VP22 protein, the PTD from the Gallid
herpesvirus 2 VP22 protein, the PTD from the Varicella-zoster virus
VP22 protein, and homologs thereof.
[0151] Examples of suitable PTDs that may be obtained or derived
from homeodomain transcription factors include, without limitation,
the homeodomain (HD) from the Drosophila Antennapedia (Antp)
protein, the HD from the Drosophila Fushi tarazu (Ftz) protein, the
HD from the Drosophila Engrailed (En) protein, the HD from the
chick Engrailed-2 protein, the HD from mammalian homeoproteins, the
HD from human homeoproteins, the HD from human Hox-A5 homeoprotein,
the HD from human Hox-A4 homeoprotein, the HD from human Hox-B5
homeoprotein, the HD from human Hox-B6 homeoprotein, the HD from
human Hox-B7 homeoprotein, the HD from human HOX-D3 homeoprotein,
the HD from human GOX homeoprotein, the HD from human MOX-2
homeoprotein, the HD from human Hoxc-8 homeoprotein, the HD from
human Islet-1 (Isl-1) homeoprotein, and homologs thereof.
[0152] Additionally, suitable PTDs include, without limitation, the
PTD derived from Kaposi-FGF (K-FGF or FGF-4), the PTD derived from
FGF-2, the PTD derived from FGF-1, and the PTD from other members
of the FGF-family of proteins.
[0153] Other suitable PTDs include synthetic PTDs (e.g., Beerens, A
M J et al. Curr Gene Ther. 2003 Oct; 3(5):486-94).
[0154] Further suitable PTDs include, without limitation, a
CHARIOT.TM. peptide (Active Motif, Carlsbad, Calif.).
[0155] In some embodiments, PTDs of the present disclosure are
produced recombinantly, while in others the PTDs are produced
synthetically or are purified from a native source.
[0156] PTD Fusion Protein Modifications
[0157] In some embodiments, PTD-containing fusion proteins of the
present disclosure include PTD-MYC fusion proteins and PTD-Bcl-2
fusion proteins that contain one or more molecules that link the
PTD to the MYC or Bcl-2 polypeptide. In some embodiments, the one
or more linker molecules are amino acid peptides.
[0158] PTD-containing fusion proteins of the present disclosure may
further contain at least one amino acid sequence that facilitates
purification of the fusion proteins. For example, the PTD-MYC
fusion proteins may contain a protein tag, such as a polyhistidine
tag, such as a six histidine epitope tag (SEQ ID NO: 6).
Alternatively, the PTD-containing fusion proteins may contain a V5
domain. Accordingly, in certain embodiments, PTD-containing fusion
proteins of the present disclosure further contain a polyhistidine
tag. Preferably, the polyhistidine tag is a 6-histidine tag (SEQ ID
NO: 6). More preferably, the histidine tag contains the sequence
HHHHHH (SEQ ID NO: 6). Additionally, the histidine tag may be added
to a PTD-containing fusion protein of the present disclosure by any
suitable method known in the art. For example, a sequence may be
cloned into an expression vector encoding a polyhistidine tag.
Alternatively, a polyhistidine tag may be added by PCR (i.e., the
PCR primers contain a polyhistidine sequence).
[0159] Moreover, a PTD-containing fusion protein of the present
disclosure may also contain at least one protein tag. In some
embodiments, the at least one protein tag is an epitope tag.
Preferably, the epitope tag is a V5 epitope tag. In some
embodiments, the V5 epitope tag contains the amino acid sequence:
GKPIPNPLLGLDST (SEQ ID NO: 7), while in other the V5 epitope tag
contains the amino acid sequence: IPNPLLGLD (SEQ ID NO: 8). The
amino acids may be either in the D formation, or in the L
formation. In some embodiments, a first plurality of amino acids is
in the D formation and a second plurality is in the L formation.
Additionally, a V5 epitope tag of the present disclosure may be
added to a PTD-containing fusion protein of the present disclosure
by any suitable method known in the art. For example, a
PTD-containing fusion protein sequence may be cloned into an
expression vector encoding a V5 epitope tag. Alternatively, a V5
epitope tag may be added by PCR (i.e., the PCR primers contain a V5
epitope sequence).
[0160] In certain embodiments, a PTD-containing fusion protein of
the present disclosure further contains a polyhistidine tag and an
epitope tag. Preferably, the PTD-containing fusion protein contains
a 6-histidine tag (SEQ ID NO: 6) and a V5 epitope tag.
[0161] In some embodiments, a PTD-containing fusion protein of the
present disclosure can be arranged in any desired order. In some
embodiments, the PTD-containing fusion protein can be arranged in
order of a) the protein transduction domain connected in frame to
the Myc or Bcl-2 polypeptide, b) the Mycor Bcl-2 polypeptide
connected in frame to the V5 domain, and c) the V5 domain connected
in frame to the six histidine epitope tag (SEQ ID NO: 6). In some
embodiments, the PTD-containing fusion protein has an order of
components of a) the Myc or Bcl-2 polypeptide connected in frame to
the protein transduction domain, b) the protein transduction domain
connected in frame to the V5 domain, and c) the V5 domain connected
in frame to the six histidine epitope tag (SEQ ID NO: 6). In some
embodiments, additional intervening amino acid sequences can be
included between each of the sequences. In some embodiments,
additional amino acid sequences can be included at the start and/or
end of the sequences.
[0162] In some embodiments, the PTD-containing fusion protein
contains a protein transduction domain, a Myc or Bcl-2 polypeptide,
and a short peptide domain. The short peptide domain can be varied.
In some embodiments, the short peptide domain is selected from at
least one of a V5, a histidine-tag, HA (hemagglutinin) tags, FLAG
tag, CBP (calmodulin binding peptide), CYD (covalent yet
dissociable NorpD peptide), StrepII, or HPC (heavy chain of protein
C). In some embodiments, the short peptide domain is about 10 or 20
amino acids long. In some embodiments, the short peptide domain is
2-20, for example 6-20 amino acids in length. In some embodiments,
two of the above listed items (for example, V5 and the his-tag) can
be used together as the short peptide domain.
[0163] Construction of PTD-Containing Fusion Proteins
[0164] PTD-containing fusion proteins of the present disclosure may
be constructed by any suitable method known in the art (e.g., U.S.
Patent Application Publication No. US 2010/0055129).
[0165] In one non-limiting example, a nucleic acid sequence
encoding a PTD-MYC fusion protein of the present disclosure may be
generated by PCR. This may be accomplished by designing a forward
primer for a MYC sequence that contains an in frame PTD sequence,
such as the RKKRRQRRR 9-amino-acid sequence of TAT ("RKKRRQRRR"
disclosed as SEQ ID NO: 5), and a reverse primer for the MYC
sequence that is designed to remove the stop codon. The PCR product
from a PCR reaction using such primers may then be cloned into any
suitable expression vector known in the art.
[0166] In one non-limiting example, a nucleic acid sequence
encoding a PTD-Bcl-2 fusion protein of the present disclosure may
be generated by PCR. This may be accomplished by designing a
forward primer for a Bcl-2 sequence that contains an in frame PTD
sequence, such as the RKKRRQRRR 9-amino-acid sequence of TAT
("RKKRRQRRR" disclosed as SEQ ID NO: 5), and a reverse primer for
the Bcl-2 sequence that is designed to remove the stop codon. The
PCR product from a PCR reaction using such primers may then be
cloned into any suitable expression vector known in the art. The
Bcl-2 unstructured loop may be removed from the BCL-2 coding
sequence using a site directed mutagenesis kit.
[0167] PTD-Containing Compositions
[0168] In other embodiments, PTD-containing fusion proteins of the
present disclosure are included in a composition. For therapeutic
methods, such fusion protein-containing compositions may include a
pharmaceutically acceptable carrier, which includes
pharmaceutically acceptable excipients and/or delivery vehicles,
for delivering a PTD-containing fusion protein to a subject, such
as an HSC transplant recipient.
[0169] In some embodiments, the PTD-containing fusion protein is a
PTD-MYC, and a therapeutically effective amount of the PTD-MYC
fusion protein-containing composition (PTD-MYC composition) is
administered to a subject to achieve an at least 50% acceleration
in hematopoietic compartment reconstitution compared to
hematopoietic compartment reconstitution in a subject that is not
administered the second composition. In other embodiments, a
therapeutically effective amount of a PTD-MYC composition is
administered to a subject to achieve enhanced hematopoietic
compartment autoreconstitution compared to hematopoietic
compartment autoreconstitution in a subject that is not
administered the PTD-MYC composition. Advantageously, PTD-MYC
compositions of the present disclosure have low toxicity when
administered to a subject. Accordingly, a PTD-MYC composition may
be administered in an amount that ranges from about 0.1 to about 50
mg/kg of the weight of the subject. In certain embodiments, a
therapeutically effective amount of a composition containing a
PTD-MYC fusion protein is at least 0.1 mg/kg, at least 0.2 mg/kg,
at least 0.3 mg/kg, at least 0.4 mg/kg, at least 0.5 mg/kg, at
least 0.6 mg/kg, at least 0.7 mg/kg, at least 0.8 mg/kg, at least
0.9 mg/kg, at least 1 mg/kg, at least 2 mg/kg, at least 3 mg/kg, at
least 4 mg/kg, at least 5 mg/kg, at least 6 mg/kg, at least 7
mg/kg, at least 8 mg/kg, at least 9 mg/kg, at least 10 mg/kg, at
least 20 mg/kg, at least 30 mg/kg, at least 40 mg/kg, or at least
50 mg/kg of the weight of the subject.
[0170] In other embodiments, the PTD-containing fusion protein is a
PTD-Bcl-2, and a therapeutically effective amount of the PTD-Bcl-2
fusion protein-containing composition (PTD-Bcl-2-composition) is
administered to a subject to achieve an at least 50% acceleration
in hematopoietic compartment reconstitution compared to
hematopoietic compartment reconstitution in a subject that is not
administered the second composition. In other embodiments, a
therapeutically effective amount of a PTD-Bcl-2-composition is
administered to a subject to achieve enhanced hematopoietic
compartment autoreconstitution compared to hematopoietic
compartment autoreconstitution in a subject that is not
administered the PTD-Bcl-2-composition. Advantageously,
PTD-Bcl-2-compositions of the present disclosure have low toxicity
when administered to a subject. Accordingly, a
PTD-Bcl-2-composition may be administered in an amount that ranges
from about 0.1 to about 50 mg/kg of the weight of the subject. In
certain embodiments, a therapeutically effective amount of a
composition containing a PTD-Bcl-2 fusion protein is at least 0.1
mg/kg, at least 0.2 mg/kg, at least 0.3 mg/kg, at least 0.4 mg/kg,
at least 0.5 mg/kg, at least 0.6 mg/kg, at least 0.7 mg/kg, at
least 0.8 mg/kg, at least 0.9 mg/kg, at least 1 mg/kg, at least 2
mg/kg, at least 3 mg/kg, at least 4 mg/kg, at least 5 mg/kg, at
least 6 mg/kg, at least 7 mg/kg, at least 8 mg/kg, at least 9
mg/kg, at least 10 mg/kg, at least 20 mg/kg, at least 30 mg/kg, at
least 40 mg/kg, or at least 50 mg/kg of the weight of the
subject.
[0171] PTD-MYC compositions of the present disclosure and/or
PTD-Bcl-2-compositions of the present disclosure can be
administered at least once a day, at least twice a day, at least
three times a day, at least four times a day, at least five times a
day, or more times a day. Alternatively, PTD-MYC compositions of
the present disclosure and/or PTD-Bcl-2-compositions of the present
disclosure can be administered at least once every two days, at
least once every three days, at least once every four days, at
least once every five days, or at least once every six days, at
least once every week, at least once every two weeks, at least once
every three weeks, or at least once every four weeks.
[0172] Advantageously, PTD-MYC compositions of the present
disclosure and/or PTD-Bcl-2-compositions of the present disclosure
can accelerate hematopoietic compartment reconstitution from HSC
transplantation (e.g., HSC engraftment), such as from bone marrow
transplantation, in a subject when administered from at least about
or about 5 days to at least about or about 12 hours before an HSC
transplant, concurrently with an HSC transplant, or from at least
about or about 12 hours to at least about or about 3 weeks after an
HSC transplant. A PTD-MYC composition of the present disclosure
and/or a PTD-Bcl-2-composition of the present disclosure may be
administered at any suitable location, including, without
limitation, the same location where the HSC transplantation occurs,
a clinic or doctor's office that is separate from the location
where the HSC transplantation occurs, and the home or residence of
the subject receiving the HSC transplantation. Any method of
administering PTD-MYC compositions of the present disclosure and/or
PTD-Bcl-2-compositions of the present disclosure known in the art
and disclosed herein may be used. In some embodiments, the PTD-MYC
composition and/or the PTD-Bcl-2-composition is administered
together with any suitable pharmaceutically acceptable carrier
known in the art and disclosed herein. In other embodiments, the
PTD-MYC composition and/or the PTD-Bcl-2-composition is
administered as a suitable formulation known in the art, including,
without limitation, an intravenous formulation.
[0173] As used herein, administering a PTD-MYC composition of the
present disclosure and/or a PTD-Bcl-2-composition of the present
disclosure occurs "concurrently" with an HSC transplantation when
it is administered either in the same mixture and/or formulation as
the HSCs to be transplanted, or in a separate mixture and/or
formulation as that of the HSCs to be transplanted. When the
PTD-MYC composition and/or the PTD-Bcl-2-composition is
administered in a separate mixture and/or formulation,
administering a PTD-MYC composition of the present disclosure
occurs and/or a PTD-Bcl-2-composition of the present disclosure
"concurrently" with an HSC transplantation includes, without
limitation, administering the PTD-MYC composition and/or the
PTD-Bcl-2-composition from at least about or about 11 hours to at
least about or about 1 minute before HSC transplantation, or from
at least about or about 1 minute to at least about or about 11
hours after HSC transplantation. As used herein, administering a
PTD-MYC composition of the present disclosure and/or a
PTD-Bcl-2-composition of the present disclosure occurs "before" an
HSC transplantation when it is administered in a separate mixture
and/or formulation as that of the HSCs to be transplanted from at
least about or about 5 days to at least about or about 12 hours
before the HSC transplantation. As used herein, administering a
PTD-MYC composition of the present disclosure occurs and/or a
PTD-Bcl-2-composition of the present disclosure "after" an HSC
transplantation when it is administered in a separate mixture
and/or formulation as that of the HSCs to be transplanted from at
least about or about 12 hours to at least about or about 3 weeks
after the HSC transplantation.
[0174] Accordingly, in certain embodiments, a PTD-MYC composition
of the present disclosure and/or a PTD-Bcl-2-composition of the
present disclosure is administered before, after, or concurrently
with an HSC transplant in a subject. In some embodiments, the
PTD-MYC composition and/or the PTD-Bcl-2-composition is
administered at least about or about 5 days, at least about or
about 4 days, at least about or about 3 days, at least about or
about 2 days, at least about or about 1 day, at least about or
about 23 hours, at least about or about 22 hours, at least about or
about 21 hours, at least about or about 20 hours, at least about or
about 19 hours, at least about or about 18 hours, at least about or
about 17 hours, at least about or about 16 hours, at least about or
about 15 hours, at least about or about 14 hours, at least about or
about 13 hours, or at least about or about 12 hours before an HSC
transplant. In other embodiments, the PTD-MYC composition and/or
the PTD-Bcl-2-composition is administered concurrently with an HSC
transplant. In further embodiments, the PTD-MYC composition and/or
the PTD-Bcl-2-composition is administered at least about or about
12 hours, at least about or about 13 hours, at least about or about
14 hours, at least about or about 15 hours, at least about or about
16 hours, at least about or about 17 hours, at least about or about
18 hours, at least about or about 19 hours, at least about or about
20 hours, at least about or about 21 hours, at least about or about
22 hours, or at least about or about 23 hours, at least about or
about 1 day, at least about or about 2 days, at least about or
about three days, at least about or about 4 days, at least about or
about 5 days, at least about or about 6 days, at least about or
about 1 week, at least about or about 2 weeks, or at least about or
about 3 weeks after an HSC transplant.
[0175] Therapeutic Uses
[0176] PTD-MYC compositions of the present disclosure and/or
PTD-Bcl-2-compositions of the present disclosure find many
therapeutic uses in the treatment of various conditions, diseases,
and syndromes. Such uses include, without limitation, accelerating
hematopoietic compartment reconstitution in an HSC transplant
recipient, such as a bone marrow transplant recipient; enhancing
hematopoietic compartment autoreconstitution; treating decreases in
the hematopoietic compartment due to chemotherapy or radiation
therapy; treating bone marrow failure syndromes; and treating
long-term HSC engraftment failure.
[0177] As used herein, a "preventing a decrease in hematopoietic
compartment cells" due to chemotherapy, radiation therapy, bone
marrow failure, and/or any other condition that leads to loss of
hematopoietic compartment cells, refers to reducing and/or
inhibiting the reduction in the amount of any hematopoietic
compartment cells of the present disclosure that are lost due to
cell necrosis, apoptosis, or the like as a result of the
chemotherapy, radiation therapy, bone marrow failure, and/or
condition that leads to loss of hematopoietic compartment cells.
Any method known in the art and disclosed herein for quantifying
cell loss (e.g., quantifying cell necrosis, apoptosis, etc.) may be
used. In one non-limiting example DNA-intercalating dyes are used
to quantify cell loss. In some embodiments, "preventing a decrease
in hematopoietic compartment cells" may include, without
limitation, an at least about or about 25% to an at least about or
about 99%, or more reduction in hematopoietic compartment cell loss
due to chemotherapy, radiation therapy, bone marrow failure, and/or
condition that leads to loss of hematopoietic compartment
cells.
[0178] In certain embodiments, PTD-MYC compositions of the present
disclosure and/or PTD-Bcl-2-compositions of the present disclosure
may be used to reduce the risk of engraftment failure and
opportunistic infections in an HSC transplant recipient by
accelerating hematopoietic compartment reconstitution in the
recipient.
[0179] Alternatively, PTD-MYC compositions of the present
disclosure and/or PTD-Bcl-2-compositions of the present disclosure
may be used to treat bone marrow failure syndromes in a subject.
Bone marrow failure syndromes can be inherited, or can occur as a
result of, for example, an infectious disease, or chronic fatigue
syndrome. Additionally, bone marrow failure in an HSC transplant
recipient can occur as result of, for example, a transplant of an
insufficient number of HSCs, a transplant of mismatched HSCs, or
the health status of the recipient.
[0180] Acquired bone marrow failure syndromes may include, without
limitation, aplastic anemia and Gulf War Syndrome.
[0181] Inherited bone marrow failure syndromes (IBMFS) may include,
without limitation, amegakaryocytic thrombocytopenia,
Diamond-Blackfan anemia, dyskeratosis congenita, fanconi anemia,
Pearson syndrome, severe congenital neutropenia, Shwachman-Diamond
syndrome, and thrombocytopenia absent radii, IVIC syndrome, WT
syndrome, radio-ulnar synostosis, and ataxia pancytopenia.
[0182] In further embodiments, PTD-MYC compositions of the present
disclosure and/or PTD-Bcl-2-compositions of the present disclosure
may be used in treating an HSC transplant recipient whose
hematopoietic chimaerism is failing after an initial engraftment of
transplanted HSCs (i.e., failure of long-term HSC engraftment).
[0183] Exposure to toxins, such as chemicals, chemotherapeutic
drugs, or radiation may lead to a decrease or loss in a subject's
hematopoietic compartment. As used herein, a decrease in the
hematopoietic compartment may occur when there is a decrease in the
total amount cells in the hematopoietic compartment or a decrease
in a specific population or sub-population of cells in the
hematopoietic compartment. Examples of specific populations or
sub-populations of cells in the hematopoietic compartment include,
without limitation, myeloid cells, monocytes, macrophages,
neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes,
platelets, dendritic cells, lymphocytes, T-cell progenitors,
pro-T-cells, pre-T-cells, double positive T-cells, immature
T-cells, mature T-cells, B-cell progenitors, pro-B-cells, early
pro-B-cells, late-pro B-cells, large pre-B-cells, small
pre-B-cells, immature B-cells, mature B-cells, NKT-cells, and
NK-cells.
[0184] Accordingly, in certain embodiments, PTD-MYC compositions of
the present disclosure and/or PTD-Bcl-2-compositions of the present
disclosure may be used to treat subjects that have been exposed to
such toxins. In some embodiments, treating subjects with a PTD-MYC
composition of the present disclosure and/or a
PTD-Bcl-2-composition of the present disclosure prevents a decrease
or loss in hematopoietic compartment cells in subjects that are
undergoing or have undergone chemotherapy or radiation therapy. In
other embodiments, treating subjects with a PTD-MYC composition of
the present disclosure and/or a PTD-Bcl-2-composition of the
present disclosure prevents a decrease or loss in the amount of
endogenous HSCs in subjects that are undergoing or have undergone
chemotherapy or radiation therapy.
Hematopoietic Stem Cells
[0185] Other aspects of the present disclosure relate to treating a
population of hematopoietic stem cells (HSCs) with a
MYC-composition of the present disclosure, a Bcl-2-composition of
the present disclosure, or both prior to transplanting the HSCs in
a subject in need thereof to enhance hematopoietic compartment
reconstitution. Other aspects of the present disclosure relate to
enhancing hematopoietic compartment cell formation in a subject in
need thereof by administering a MYC-composition of the present
disclosure, a Bcl-2-composition of the present disclosure, or both
to induce HSCs to generate hematopoietic compartment cells. A
"subject", "patient", or "host" to be treated by the methods of the
present disclosure may be a human or non-human such as any of the
non-human animal disclosed herein, in need of hematopoietic
compartment cell formation (e.g., hematopoietic compartment
reconstitution or autoreconstitution) enhancement. As disclosed
herein HSCs of the present disclosure pre-treated with a
MYC-composition of the present disclosure, a Bcl-2-composition of
the present disclosure, or both may be washed prior to being
administered to a subject in need of hematopoietic compartment
reconstitution. Alternatively, the pre-treated HSCs may be
administered to the subject without being washed prior to
administration as the transient effects of the MYC-composition
and/or Bcl-2-composition are not harmful to the subject. Any
suitable solution known in the art as disclosed herein may be used
to wash the pre-treated HSCs. In one non-limiting example, a
pH-balanced saline solution is used to wash the pre-treated HSCs
prior to administering the HSCs to the subject. The pre-treated
HSCs may be washed from less than about or about 1 minute to about
1 hour, about 4 hours, about 6 hours, about 8 hours, about 12
hours, about 24 hours or more prior administering to the
subject.
[0186] HSCs of the present disclosure are able to give rise to all
cell types of the hematopoietic compartment, including, without
limitation the myeloid lineage, which includes, without limitation,
monocytes, macrophages, neutrophils, basophils, eosinophils,
erythrocytes, megakaryocytes, platelets, and dendritic cells; and
the lymphoid lineage, which includes, without limitation, T-cells,
B-cells, NKT-cells, and NK cells.
[0187] In some embodiments, a MYC-composition of the present
disclosure accelerates hematopoietic compartment reconstitution in
HSC transplant recipients. As disclosed herein, HSC transplant
recipients may receive a bone marrow transplant, an HSC-enriched
bone marrow transplant, a transplant of cord blood, a transplant of
HSC-enriched cord blood, a transplant of placenta-derived blood, a
transplant of purified or partially-purified HSCs, a transplant of
HSCs derived from an HSC cell line, or a transplant of
conditionally immortalized HSCs. In such embodiments, HSCs may be
administered as a step in the process of an HSC transplantation
procedure. As disclosed herein, the HSCs may be included, without
limitation, in transplanted bone marrow, transplanted cord blood,
or in transplanted cell lines. In certain preferred embodiments,
the transplant recipient is a human subject. Suitable HSCs may be
obtained by any suitable technique known in the art. For example,
HSCs may be found in the bone marrow of a donor, which includes
femurs, hip, ribs, sternum, and other bones. Any method known in
the art for extracting or harvesting bone marrow cells may be used.
In one non-limiting example, HSCs may be obtained directly from the
marrow cavity of the hip using a needle and syringe to aspirate
cells from the marrow cavity. Rich marrow may be obtained from the
hip by performing multiple small aspirations.
[0188] HSCs suitable for use with the methods of the present
disclosure may be produced from embryonic stem (ES) cells and/or
induced pluripotent stem (iPS) cells. Any method of producing HSCs
from ES cells and/or iPS cells known in the art may be used (e.g.,
Keller, G. Genes Dev. 2005 19: 1129-1155; and Papapetrou Sadelain,
F1000 Med Rep. 2010 Jun. 16; 2). For example, HSCs may be produced
from ES cells by patterning the hematopoietic development of ES
cell culture on the hematopoietic commitment in the early embryo
(e.g., Keller, G. Genes Dev. 2005 19: 1129-1155).
[0189] Additionally, HSCs suitable for use with the methods of the
present disclosure may be obtained by any suitable technique known
in the art. For example, HSCs may be found in the bone marrow of a
donor, which includes femurs, hip, ribs, sternum, and other bones.
Any method known in the art for extracting or harvesting bone
marrow cells may be used. In one non-limiting example, HSCs may be
obtained directly from the marrow cavity of the hip using a needle
and syringe to aspirate cells from the marrow cavity. Rich marrow
may be obtained from the hip by performing multiple small
aspirations.
[0190] Suitable HSCs may also be obtained from peripheral blood
cells found in the blood of a donor, often following pre-treatment
with cytokines, such as G-CSF (granulocyte colony-stimulating
factors), that induce HSCs to be released from the bone marrow
compartment of the donor. HSCs may also be obtained from peripheral
blood that has undergone an apheresis procedure to enrich for HSC.
Any apheresis procedure known in the art may be used. In certain
embodiments, the apheresis procedure is a leukapheresis
procedure.
[0191] Additionally, suitable HSCs may be obtained from umbilical
cord blood, placenta, and mobilized peripheral blood. For
experimental purposes, fetal liver, fetal spleen, and AGM
(Aorta-gonad-mesonephros) of animals are also useful sources of
HSCs. Additionally, HSCs may be procured from a source that
obtained HSCs from the bone marrow, peripheral blood, umbilical
cord, or fetal tissue of a donor. Alternatively, the HSCs may be
included in a bone marrow, peripheral blood, umbilical cord, or
fetal tissue sample of a donor.
[0192] In some embodiments, HSCs are obtained from a human
umbilical cord or placenta. Another source of HSCs that may be
utilized is the developing blood-producing tissues of fetal
animals. In humans, HSCs may be found in the circulating blood of a
human fetus by about 12 to 18 weeks. In some embodiments, human
HSCs are obtained from any source, e.g., the bone marrow, umbilical
cord, peripheral blood, or fetal tissue of blood, of type A+, A-,
B+, B-, O+, O-, AB+, and AB-donors. In other embodiments, human
HSCs are obtained from any source, e.g., the bone marrow, umbilical
cord, peripheral blood, or fetal tissue of blood, of universal
donors or donors having a rare blood type. Rare blood types are
known in the art and include, without limitation, Oh, CDE/CDE,
CdE/CdE, C.sup.wD-/C.sup.wD-, -D-/-D-, Rh.sub.null, Rh:-51,
LW(a-b+), LW(a-b-), S-s-U-, S-s-U(+), pp, Pk, Lu(a+b-), Lu(a-b-),
Kp(a+b-), Kp(a-b-), Js(a+b-), Ko, K:-11, Fy(a-b-), Jk(a-b-),
Di(b-), I-, Yt(a-), Sc:-1, Co(a-), Co(a-b-), Do(a-), Vel-, Ge-,
Lan-, Lan(+), Gy(a-), Hy-, At(a-), Jr(a-), In(b-), Tc(a-), Cr(a-),
Er(a-), Ok(a-), JMH-, and En(a-).
[0193] In other embodiments, human HSCs are obtained from any
source, e.g., the bone marrow, umbilical cord, peripheral blood, or
fetal tissue of blood, of donors having an autoimmune disorder,
immune deficiency, or any other disease or disorder that would
benefit from a transplantation of HSCs. Such donors may also be the
recipients. Advantageously, HSCs obtained from such donor may be
used for personalized HSC therapy.
[0194] In one non-limiting example, human HSCs may be obtained by
anesthetizing the stem cell donor, puncturing the posterior
superior iliac crest with a needle, and performing aspiration of
bone marrow cells with a syringe. In another non-limiting example,
HSCs may be obtained from the peripheral blood of a donor, where a
few days prior to harvesting the stem cells form the peripheral
blood, the donor is injected with G-CSF in order to mobilize the
stem cells to the peripheral blood.
[0195] Accordingly, in some embodiments, HSCs are obtained from an
autologous donor, that is the donor will also be the recipient of
the HSCs derived from such HSCs. Any methods known in the art and
described herein may be used to obtain HSCs from the autologous
donor. The HSCs and/or any therapeutic products derived or produced
therefrom are then transplanted, administered, and or transfused
back to the original donor. Similarly, HSCs may be obtained from an
allogenic donor, such as a sibling, parent, or other relative of a
subject in need of an HSC transplantation. In one non-limiting
example, allogenic HSCs are obtained by collecting HSCs from
different blood groups or major histocompatibility complex (MHC) or
human leukocyte antigen (HLA) matching sources. Autologous and/or
allogenic HSC transplantation may occur at any time after the
donation, such as days later, months later, or even years later.
Autologous donation may be particularly useful in cases where the
subject in need of HSCs would have a negative, deleterious, or
toxic reaction to transplantation and/or transfusion of HSCs from
any other donor, including allogenic and/or universal donors.
Examples of patients that may benefit from autologous and/or
allogenic donation are well known in the art and include, without
limitation, those suffering from an autoimmune disorder, blood
disease or disorder, immune disease or disorder, or other related
diseases or conditions.
[0196] Cells obtained from, for example, bone marrow, peripheral
blood, or cord blood, are typically processed after extraction or
harvest. Any method known in the art for processing extracted or
harvest cells may be used. Examples of processing steps include,
without limitation, filtration, centrifugation, screening for
hematopathologies, screening for viral and/or microbial infection,
erythrocyte depletion, T-cell depletion to reduce incidence of
graft-versus-host disease in allogenic stem cell transplant
recipients, volume reduction, cell separation, resuspension of
cells in culture medium or a buffer suitable for subsequent
processing, separation of stem cells from non-stem cells e.g., stem
cell enrichment), ex vivo or in vitro stem cell expansion with
growth factors, cytokines, and/or hormones, and
cryopreservation.
[0197] Any suitable method for stem cell enrichment known in the
art may be used. Examples of stem cell enrichment methods include,
without limitation, fluorescence activated cell sorting (FACS) and
magnetic activated cell sorting (MACS).
[0198] Accordingly, in certain embodiments, HSCs suitable for use
in the methods of the present disclosure are human HSCs. In other
embodiments, HSCs suitable for use in the methods of the present
disclosure are autologous to the subject. In further embodiments,
HSCs suitable for use in the methods of the present disclosure are
allogenic to the subject.
[0199] HSCs obtained from a donor may be identified and/or enriched
by any suitable method of stem cell identification and enrichment
known in the art, such as by utilizing certain phenotypic or
genotypic markers. For example, in some embodiments, identification
of HSCs includes using cell surface markers associated with HSCs or
specifically associated with terminally differentiated cells of the
system. Suitable surface markers may include, without limitation,
one or more of c-kit, Sca-1, CD4, CD34, CD38, Thy1, CD2, CD3, CD4,
CD5, CD8, CD43, CD45, CD59, CD90, CD105, CD133, CD135, ABCG2,
NK1.1, B220, Ter-119, Flk-2, CDCP1, Endomucin, Gr-1, CD46, Mac-1,
Thy1.1, and the signaling lymphocyte activation molecule (SLAM)
family of receptors. Examples of SLAM receptors include, without
limitation, CD150, CD48, and CD244.
[0200] Additionally, HSCs obtained from a donor may be separated
from non-stem cells by any suitable method known in the art
including, without limitation, fluorescence activated cell sorting
(FACS) and magnetic activated cell sorting (MACS).
[0201] In one non-limiting example, human peripheral blood cells
are incubated with antibodies recognizing c-kit, Sca-1, CD34, CD38,
Thy1, CD2, CD3, CD4, CD5, CD8, CD43, CD45, CD59, CD90, CD105,
CD133, ABCG2, NK1.1, B220, Ter-119, Flk-2, CDCP1, Endomucin, or
Gr-1. Antibodies for CD2, CD3, CD4, CD5, CD8, NK1.1, B220, Ter-119,
and Gr-1 are conjugated with magnetic beads. The cells expressing
CD2, CD3, CD4, CD5, CD8, NK1.1, B220, Ter-119, or Gr-1 are retained
in the column equipped to trap magnetic beads and cells attached to
magnetic bead conjugated antibodies. The cells that are not
captured by the MACS column are subjected to FACS analysis.
Antibodies for c-kit, Sca-1, CD34, CD38, Thy1, are conjugated with
fluorescent materials known in the art. The cells that are CD34+,
CD38.sup.low/-, c-kit.sup.-/low, Thy1.sup.+ are separated from the
rest of sample by virtue of the types of fluorescent antibodies
associated with the cells. These cells are provided as human
long-term HSCs suitable for use with any of the methods of the
present disclosure.
[0202] In another non-limiting example, cells obtained from a
subject are labeled with the same set of magnetic bead conjugated
antibodies as described above (antibodies against one or more of
CD2, CD3, CD4, CD5, CD8, NK1.1, B220, Ter-119, or Gr-1) and
fluorescent conjugated CD150, CD244 and/or CD48 antibodies. After
removing cells captured by the magnetic bead conjugated antibodies
from the sample, the sample is analyzed by FACS and CD150.sup.+,
CD244.sup.- and CD48.sup.- cells are retained as long-term
HSCs.
[0203] In some embodiments, HSCs utilized in the methods of the
present disclosure contain one or more of the markers: c-kit.sup.+,
Sca-1.sup.+, CD34.sup.low/-, CD38.sup.+, Thy1.sup.+/low,
CD34.sup.+, CD38.sup.low/-, c-kit.sup.-/low, and/or Thy1.sup.+. In
some embodiments, the HSCs utilized in the methods of the present
disclosure lack one or more of the markers: CD2, CD3, CD4, CD5,
CD8, NK1.1, B220, Ter-119, and/or Gr-1. In certain embodiments, the
HSCs utilized in the methods of the present disclosure are of an
A.sup.+, A.sup.-, B.sup.+, B.sup.-, O.sup.+, O.sup.-, AB.sup.+, or
AB.sup.- type.
[0204] Alternatively, suitable HSCs may be obtained from a
non-human source. Suitable non-human HSCs may be isolated from,
femurs, hip, ribs, sternum, and other bones of a non-human animal,
including, without limitation, laboratory/research animals,
rodents, pets, livestock, farm animals, work animals, pack animals,
rare or endangered species, racing animals, and zoo animals.
Further examples of suitable non-human animals include, without
limitation, monkeys, primates, mice, rats, guinea pigs, hamsters,
dogs, cats, horses, cows, pigs, sheep, goats, and chickens. For
example, HSCs may be obtained from murine bone marrow cells, by
incubating the bone marrow cells with antibodies recognizing cell
surface molecules such as one or more of c-kit, Sca-1, CD34, CD38,
Thy1, CD2, CD3, CD4, CD5, CD8, CD43, CD45, CD59, CD90, CD105,
CD133, ABCG2, NK1.1, B220, Ter-119, Flk-2, CDCP1, Endomucin, or
Gr-1. Antibodies for CD2, CD3, CD4, CD5, CDS, NK1.I, B220, Ter-119,
and Gr-1 are conjugated with magnetic beads. In MACS equipment, the
cells harboring CD2, CD3, CD4, CD5, CD8, NK1.1, B220, Ter-119, or
Gr-1 on their surface are retained in the column equipped to trap
magnetic beads and the cells attached to magnetic bead conjugated
antibodies. The cells that are not captured by MACS column are
subjected to FACS analysis. For FACS analysis, Antibodies for
surface molecules such as c-kit, Sca-1, CD34, CD38, Thy1, are
conjugated with fluorescent materials. The cells that are
c-kit.sup.+, Sca-1.sup.+, CD34.sup.low/-, CD38.sup.+,
Thy1.sup.+/low are separated from the rest of the sample by virtue
of the types of fluorescent antibodies associated with the cells.
These cells are provided as murine long-term HSCs suitable for use
with any of the methods of the present disclosure. In other
embodiments, different sets of marker are used to separate murine
long-term HSCs from cells of bone marrow, umbilical cord blood,
fetal tissue, and peripheral blood.
[0205] In some embodiments, obtaining HSCs from bone marrow
includes first injecting the HSC donor, such as a mouse, with
5-fluorouracil (5-FU) to induce the HSCs to proliferate in order to
enrich for HSCs in the bone marrow of the donor.
[0206] Moreover, HSCs suitable for use with any of the methods of
the present disclosure, whether obtained from, or present in cord
blood, bone marrow, peripheral blood, or other source, may be grown
or expanded in any suitable, commercially available or custom
defined medium, with or without serum, as desired (e.g., Hartshorn
et al., Cell Technology for Cell Products, pages 221-224, R. Smith,
Editor; Springer Netherlands, 2007). For example, serum free medium
may utilize albumin and/or transferrin, which have been shown to be
useful for the growth and expansion of CD34.sup.+ cells in serum
free medium. Also, cytokines may be included, such as Flt-3 ligand,
stem cell factor (SCF), and thrombopoietin (TPO), among others.
HSCs may also be grown in vessels such as bioreactors (e.g., Liu et
al., Journal of Biotechnology 124:592-601, 2006). A suitable medium
for ex vivo expansion of HSCs may also contain HSC supporting
cells, such as stromal cells (e.g., lymphoreticular stromal cells),
which can be derived, for example, from the disaggregation of
lymphoid tissue, and which have been shown to support the in vitro,
ex vivo, and in vivo maintenance, growth, and differentiation of
HSCs, as well as their progeny.
[0207] HSC growth or expansion may be measured in vitro or in vivo
according to routine techniques known in the art. For example, WO
2008/073748, describes methods for measuring in vivo and in vitro
expansion of HSCs, and for distinguishing between the
growth/expansion of HSCs and the growth/expansion of other cells in
a potentially heterogeneous population (e.g., bone marrow), such as
intermediate progenitor cells.
[0208] HSC Cell Lines
[0209] In other embodiments, HSCs suitable for use in any of the
methods of the present disclosure may also be derived from an HSC
cell line. Suitable HSC cell lines include any cultured
hematopoietic stem cell line known in the art. Non-limiting
examples include the conditionally immortalized long-term stem cell
lines described in U.S. Patent Application Publication Nos. US
2007/0116691 and US 2010/0047217.
[0210] In certain embodiments, HSCs suitable for use in the methods
of the present disclosure are conditionally immortalized before
administering the HSCs to a subject. For example, HSCs obtained by
any method disclosed herein may be treated with a regulatable
(e.g., inducible, controllable) protooncogene that promotes cell
survival and proliferation, such as MYC, and/or with a protein that
inhibits apoptosis of the HSCs, such as Bcl-2 (e.g., U.S. Patent
Application Publication No. US 2007/0116691). In some embodiments,
the regulatable protooncogene is a MYC-composition. Preferably, the
MYC-composition is a TAT-MYC fusion protein. The protein that
inhibits apoptosis of the HSCs may also be regulatable. For
example, the regulatable protein may be a PTD-Bcl-2 fusion protein,
such as a TAT-Bcl-2 fusion protein.
[0211] In other embodiments, HSCs suitable for use in the methods
of the present disclosure are cultured in the presence of a
MYC-composition (e.g., a TAT-MYC protein fusion) of the present
disclosure, a Bcl-2-composition (e.g., a TAT-BCl-2 protein fusion)
of the present disclosure, or both before administering the HSCs to
a subject. Preferably, culturing the first composition in the
presence of the MYC-composition and/or Bcl-2-composition
conditionally immortalizes the HSCs, which results in an expansion
of the cultured HSCs.
[0212] As used herein, an "expansion of hematopoietic stem cells"
or an "expansion of HSCs" refers to an increase in cell
proliferation and/or cell survival, as compared to HSCs that have
not been cultured or treated with a MYC-composition of the present
disclosure, a Bcl-2-composition of the present disclosure, or both.
For example, HSC expansion occurs when HSC proliferation, HSC
survival, or both is increased by at least about or about 10% to at
least about or about 500%. Any method known in the art and
disclosed herein for measuring an increase in HSC proliferation
and/or survival may be used.
[0213] Accordingly, HSCs suitable for use in any of the methods of
the present disclosure may be obtained from bone marrow, from an
apheresis procedure, from peripheral blood cells, from peripheral
blood cells that have undergone leukapheresis, from umbilical cord
blood, from amniotic fluid, from cultured HSC cells, from an
immortalized HSC cell line, or from a conditionally immortalized
HSC cell line. Alternatively, HSCs suitable for use in any of the
methods of the present disclosure may be present in bone marrow, in
peripheral blood cells, in peripheral blood cells that have
undergone leukapheresis, in umbilical cord blood, in amniotic
fluid, and in cell lines.
[0214] Transgenic Approach
[0215] In some embodiments, conditionally immortalized HSCs for use
in the methods of the present disclosure are established using any
transgenic approach known in the art (e.g., U.S. Patent Application
Publication Nos. US 2007/0116691 and US 2010/0047217. For example,
HSCs may be immortalized by obtaining an expanded population of
HSCs, transfecting (transducing) the HSCs with a vector that
encodes, for example, a MYC polypeptide and/or a Bcl-2 polypeptide
(e.g., inducible and/or controllable), transfecting (transducing)
the HSCs with a vector encoding the MYC polypeptide and/or the
Bcl-2 polypeptide, and expanding the transfected HSCs in the
presence of a combination of stem cell growth factors under
conditions where the MYC polypeptide and/or Bcl-2 polypeptide is
induced and/or active.
[0216] The MYC polypeptide and/or Bcl-2 polypeptide is regulatable
(e.g., inducible or controllable), so that the polypeptide can be
activated and deactivated (i.e., turned on or turned off) as
desired to either maintain the HSCs in an immortalized state or to
allow it to differentiate into a desired cell type.
[0217] In some embodiments, the MYC polypeptide and/or Bcl-2
polypeptide has been modified such that activity is inducible or
repressible. For example, the MYC polypeptide and/or Bcl-2
polypeptide may further contain an inducible receptor. In certain
embodiments, the recombinant proteins contain an estrogen receptor
(ER). In certain embodiments, the recombinant protein that promotes
cell survival and/or proliferation and that contains an estrogen
receptor is a MYC-ER polypeptide and/or a Bcl-2-ER polypeptide. In
certain embodiments, the proteins containing an estrogen receptor
are induced by 4-hydroxytamoxifen (4-OHT). Alternatively, the
proteins may contain a glucocorticoid receptor (GR), e.g., a
glucocorticoid receptor that is sensitive to mifepristone
(MIFEPREX). In certain embodiments, the protein that contains a
glucocorticoid receptor is a MYC-GR polypeptide and/or a Bcl-2-GR
polypeptide.
[0218] Any method known in the art for obtaining an expanded
population of HSCs known in the art may be used. For example, HSCs
may be cultured with one or more growth factor that promotes cell
proliferation and/or cell division.
[0219] Preferably, the vectors are an integrating vector, which has
the ability to integrate into the genome of a cell (e.g., a
retroviral vector). The HSCs can be transfected and/or transduce
with the vectors using any suitable method of transfecting cells,
and particularly mammalian cells, including by using combinations
of techniques. Examples of suitable vectors, include without
limitation, retroviral vectors, lentivirus vectors, parvovirus
vectors, vaccinia virus vectors, coronavirus vectors, calicivirus
vectors, papilloma virus vectors, flavivirus vectors,
orthomixovirus vectors, togavirus vectors, picornavirus vectors,
adenoviral vectors, and modified and attenuated herpesviruses
vectors. Any such virus vector can further be modified with
specific surface expressed molecules that target these to HSCs,
such as membrane bound SCF, or other stem-cell specific growth
factor ligands. Other methods of transfection of mammalian cells
include, but are not limited to, direct electroporation of
mammalian expression vectors, such as by using NUCLEOFECTOR.TM.
technology (AMAXA Biosystems). This technology is a highly
efficient non-viral gene transfer method for most primary cells and
for hard-to-transfect cell lines, which is an improvement on the
long-known method of electroporation, based on the use of cell-type
specific combinations of electrical current and solutions to
transfer polyanionic macromolecules directly into the nucleus.
Additionally, suitable methods of transfection can include any
bacterial, yeast, or other artificial methods of gene delivery that
are known in the art.
[0220] Enhancement of Endogenous Expression
[0221] In some embodiments, conditionally immortalized HSCs for use
in the methods of the present disclosure may be established by
enhancing the expression of endogenous proteins that promote cell
survival and/or proliferation, including, without limitation, any
MYC protein of the present disclosure. Additionally, conditionally
immortalized HSCs for use in the methods of the present disclosure
may be established by also enhancing the expression of endogenous
proteins that inhibit apoptosis, including, without limitation, any
Bcl-2 protein of the present disclosure.
[0222] Protein Transduction Approach
[0223] In some embodiments, HSCs obtained and/or produced by any
method disclosed herein may be treated with a gene product that
promotes cell survival and/or proliferation, including, but not
limited to any MYC protein of the present disclosure, and/or with a
protein that inhibits apoptosis of the HSCs, including, but limited
to a Bcl-2 protein of the present disclosure. In some embodiments,
the MYC protein is a fusion protein containing a PTD. In some
embodiments, the Bcl-2 protein is a fusion protein containing a
PTD. In some embodiments, HSCs obtained and/or produced by any
method disclosed herein may be treated with one or more compound
(optionally an exogenous protein) that enables the transient
upregulation of at least one function of a MYC protein of the
present disclosure. In some embodiments, the MYC protein is a
PTD-MYC fusion protein. In certain embodiments, the PTD-MYC fusion
protein is a TAT-MYC fusion protein.
[0224] In some embodiments, HSCs obtained by any method disclosed
herein may be treated with one or more compound (optionally an
exogenous protein) that enables the transient upregulation of at
least one function of a Bcl-2 protein of the present disclosure. In
some embodiments, the exogenous Bcl-2 protein is a PTD-Bcl-2 fusion
protein. In some embodiments, the PTD-Bcl-2 fusion protein is a
TAT-Bcl-2 fusion protein.
[0225] In other embodiments, HSCs suitable for use in any of the
methods of the present disclosure are treated with a composition
containing a fusion protein containing a MYC protein of the present
disclosure fused to a PTD (e.g., a PTD-MYC fusion protein), a
composition containing a fusion protein containing a Bcl-2 protein
of the present disclosure fused to a PTD (e.g., a PTD-Bcl-2 fusion
protein), or both.
[0226] Accordingly, HSCs suitable for use in any of the methods of
the present disclosure may be obtained from embryonic stem cells
(ES cells), fetal stem cells, induced pluripotent stem cells (iPS
cells), bone marrow, from peripheral blood cells, from peripheral
blood cells that have undergone apheresis, from peripheral blood
cells that have undergone leukapheresis, from umbilical cord blood,
from amniotic fluid, from cultured HSC cells, from an immortalized
HSC cell line, or from a conditionally immortalized HSC cell
line.
[0227] HSC Compositions
[0228] In other embodiments, HSCs suitable for use in any of the
methods of the present disclosure are included in a composition.
Suitable HSC-containing compositions may contain, without
limitation, isolated and/or purified HSCs; whole bone marrow; bone
marrow enriched for HSCs (e.g., 5-FU treated bone marrow);
peripheral blood; and umbilical cord blood. In some embodiments,
such compositions include a pharmaceutically acceptable carrier.
Suitable pharmaceutically acceptable carriers may include
pharmaceutically acceptable excipients and/or delivery vehicles,
for delivering the HSCs to a subject, such as a patient.
Additionally, pharmaceutically acceptable carriers may contain a
cell culture medium that supports HSC viability. The medium will
generally be serum-free in order to avoid provoking an immune
response in the recipient. The carrier will generally be buffered
and/or pyrogen-free.
[0229] In some embodiments, a therapeutically effective amount of a
composition containing HSCs is administered to a subject to achieve
hematopoietic compartment reconstitution in the subject. Generally,
administering 10.sup.4 to 10.sup.6 HSCs is sufficient to achieve
hematopoietic compartment reconstitution. In certain embodiments,
administering a MYC-composition of the present disclosure, a
Bcl-2-composition of the present disclosure, or both to an HSC
transplant recipient can reduce the number of HSCs required to
achieve hematopoietic compartment reconstitution. For example,
administering a therapeutically effective amount of a
MYC-composition of the present disclosure, a Bcl-2-composition of
the present disclosure, or both to an HSC transplant recipient can
reduce the therapeutically effective amount of HSCs in an HSC
composition of the present disclosure required to achieve
hematopoietic compartment reconstitution by at least about or about
10% to least about or about 75%, compared to the amount of HSCs
required to achieve hematopoietic compartment reconstitution in an
HSC transplant recipient that is not administered the
MYC-composition and/or Bcl-2-composition.
[0230] In certain embodiments, administering a therapeutically
effective amount of a MYC-composition of the present disclosure, a
Bcl-2-composition of the present disclosure, or both to an HSC
transplant recipient can reduce the therapeutically effective
amount of HSCs in an HSC composition of the present disclosure
required to achieve hematopoietic compartment reconstitution by at
least about or about 10% to at least about or about 75%, for
example, by at least about or about 10%, at least about or about
15%, at least about or about 20%, at least about or about 25%, at
least about or about 30%, at least about or about 35%, at least
about or about 40%, at least about or about 50%, at least about or
about 55%, at least about or about 60%, at least about or about
65%, at least about or about 70%, at least about or about 75%, or a
higher percentage less, as compared to the amount of HSCs required
to achieve hematopoietic compartment reconstitution in an HSC
transplant recipient that is not administered the MYC-composition
and/or Bcl-2-composition.
[0231] In certain embodiments, treating a population with a
therapeutically effective amount of a MYC-composition, a
Bcl-2-composition, or both to a population of HSCs prior to the
HSCs being transplanted to subject in need thereof can reduce the
therapeutically effective amount of HSCs required to achieve
hematopoietic compartment reconstitution by at least about or about
10% to at least about or about 75%, for example, by at least about
or about 10%, at least about or about 15%, at least about or about
20%, at least about or about 25%, at least about or about 30%, at
least about or about 35%, at least about or about 40%, at least
about or about 50%, at least about or about 55%, at least about or
about 60%, at least about or about 65%, at least about or about
70%, at least about or about 75%, or a higher percentage less, as
compared to the amount of HSCs required to achieve hematopoietic
compartment reconstitution in an HSC transplant recipient that does
receive HSCs that were pre-treated with a MYC-composition of the
present disclosure, a Bcl-2-composition of the present disclosure,
or both.
[0232] HSC compositions and populations of HSCs of the present
disclosure are generally administered into the body of a subject,
such as a patient, by any suitable methods known in the art,
including without limitation, injection and implantation. For
example, HSC may be directly injected into the tissue in which they
are intended to act using a syringe containing an HSC composition
of the present disclosure. Alternatively, an HSC composition of the
present disclosure may be delivered via a catheter, such as a
central venous catheter, attached to a syringe containing the HSC
composition.
[0233] HSC Transplantation
[0234] In further embodiments, HSC-containing compositions and
populations of HSCs of the present disclosure are administered to a
subject in need of hematopoietic stem cell (HSC) transplantation as
a step in the process of an HSC transplantation procedure. In
certain preferred embodiments, the subject is a human patient in
need of a HSC transplant. In other embodiments, the subject is any
non-human animal, including, without limitation,
laboratory/research animals, rodents, pets, livestock, farm
animals, work animals, pack animals, rare or endangered species,
racing animals, zoo animals, monkeys, primates, mice, rats, guinea
pigs, hamsters, dogs, cats, horses, cows, pigs, sheep, goats, and
chickens.
[0235] The HSC transplantation procedure may be a myeloablative HSC
transplant.
[0236] Myeloablation generally refers to the ablation or
suppression of the endogenous hematopoietic compartment of an HSC
transplant recipient. Myeloablation occurs prior to HSC
transplantation. In HSC transplant recipients suffering from a
hematological disease, such as a hematological cancer,
myeloablation may be performed to help eradicate the disease.
Myeloablation may also be performed to suppress the endogenous
immune system of the HSC transplant recipient in order to help
reduce the risk of rejection of the transplanted HSCs (e.g.,
graft-versus-host disease). Any method known in the art for
myeloablation may be used. Examples of myeloablation procedures
include, without limitation, chemotherapy, irradiation, and
combinations thereof.
[0237] Alternatively, the HSC transplantation procedure may be
non-myeloablative. In non-myeloablative procedures lower doses of
chemotherapy and/or radiation are used in the recipient prior to
HSC transplantation.
[0238] Subjects in need of an HSC transplant include subjects
presenting with an HSC transplant indication. Examples of HSC
transplant indications include, without limitation, a hematological
malignancy, a myeloma, multiple myeloma, a leukemia, acute
lymphoblastic leukemia, chronic lymphocytic leukemia, a lymphoma,
indolent lymphoma, non-Hodgkin lymphoma, diffuse B cell lymphoma,
follicular lymphoma, mantle cell lymphoma, T cell lymphoma, Hodgkin
lymphoma, a neuroblastoma, a retinoblastoma, Shwachman Diamond
syndrome, a brain tumor, Ewing's Sarcoma, a Desmoplastic small
round cell tumor, a relapsed germ cell tumor, a hematological
disorder, a hemoglobinopathy, an autoimmune disorder, juvenile
idiopathic arthritis, systemic lupus erythematosus, severe combined
immunodeficiency, congenital neutropenia with defective stem cells,
severe aplastic anemia, a sickle-cell disease, a myelodysplastic
syndrome, chronic granulomatous disease, a metabolic disorder,
Hurler syndrome, Gaucher disease, osteopetrosis, malignant
infantile osteopetrosis, heart disease, HIV, and AIDS.
Additionally, a subject in need of an HSC transplant can also
include a subject that has had an organ transplant.
Enhancing Hematopoietic Compartment Cell Formation
[0239] Other aspects of the present disclosure relate to enhancing
hematopoietic compartment cell formation in a subject in need
thereof by administering a MYC-composition of the present
disclosure, a Bcl-2-composition of the present disclosure, or both;
and/or administering a population of HSCs that has been pre-treated
with a MYC-composition of the present disclosure, a
Bcl-2-composition of the present disclosure, or both. In some
embodiments, enhancing hematopoietic compartment cell formation
includes enhancing hematopoietic compartment reconstitution. In
some embodiments, enhancing hematopoietic compartment cell
formation includes enhancing hematopoietic compartment
autoreconstitution.
[0240] Enhancing Hematopoietic Compartment Reconstitution
[0241] In certain embodiments, administering a population of HSCs
that have been pre-treated with a MYC-composition of the present
disclosure, a Bcl-2-composition of the present disclosure, or both,
to a subject in need of HSC transplantation enhances hematopoietic
compartment reconstitution in the subject. In other embodiments,
administering a MYC-composition of the present disclosure, a
Bcl-2-composition of the present disclosure, or both to a subject
that has received or will receive an HSC transplant enhances
hematopoietic compartment reconstitution in the subject. For
example, pre-treating HSCs with the MYC-composition, the
Bcl-2-composition, or both, and then administering the HSCs; or
administering the MYC-composition, the Bcl-2-composition, or both
may improve the rate of hematopoietic compartment reconstitution,
increase the amount of hematopoietic compartment cells that are
formed, or reduce loss of hematopoietic compartment cells in the
subject, as compared to a subject that is administered HSCs that
were not pre-treated with the MYC-composition, the
Bcl-2-composition, or both; or was not administered the
MYC-composition, the Bcl-2-composition, or both.
[0242] Preferably, pre-treating the HSCs with the MYC-composition,
the Bcl-2-composition, or both prior to administering the HSCs to a
subject; or administering the MYC-composition, the
Bcl-2-composition, or both to the subject accelerates (i.e.,
increases the rate of) hematopoietic compartment reconstitution in
the subject, as compared to hematopoietic compartment
reconstitution in a subject that has not been administered the
pre-treated HSCs or the MYC-composition, the Bcl-2-composition, or
both.
[0243] As used herein, an "acceleration in hematopoietic
compartment reconstitution" refers to a reduction in the time
required to reconstitute at least about or about 25% to about 100%
of one or more of the blood cell lineages in the hematopoietic
compartment, including, without limitation, monocytes, macrophages,
neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes,
platelets, and dendritic cells; and the lymphoid lineage, which
includes, without limitation, T-cells, B-cells, NKT-cells, and NK
cells. For example, a reduction from 8 weeks to 4 weeks in the time
required to reconstitute the hematopoietic compartment would
constitute an acceleration of 50%.
[0244] Pre-treatment with a MYC-composition of the present
disclosure, a Bcl-2-composition of the present disclosure, or both;
or administration of a MYC-composition of the present disclosure, a
Bcl-2-composition of the present disclosure, or both may achieve an
at least about or about 25% to art least about or about 500%, for
example, an at least about or about 25%, at least about or about
30%, at least about or about 31%, at least about or about or about
32%, at least about or about 33%, at least about or about 34%, at
least about or about 35%, at least about or about 40%, at least
about or about 45%, at least about or about 50%, at least about or
about 55%, at least about or about 60%, at least about or about
65%, at least about or about 66%, at least about or about 67%, at
least about or about 68%, at least about or about 69%, at least
about or about 70%, at least about or about 75%, at least about or
about 80%, at least about or about 90%, at least about or about
95%, at least about or about 100%, at least about or about 150%, at
least about or about 200%, at least about or about 250%, at least
about or about 300%, at least about or about 400%, at least about
or about 500%, or a higher percentage acceleration in hematopoietic
compartment reconstitution in a subject that has received or will
receive an HSC transplant, compared to hematopoietic compartment
reconstitution in a subject that is not administered the
pre-treated HSCs; or the MYC-composition, the Bcl-2-composition, or
both.
[0245] As disclosed herein, hematopoietic compartment
reconstitution includes, without limitation, T-cell compartment
reconstitution, B-cell compartment reconstitution, NK-cell
compartment reconstitution, myeloid cell compartment
reconstitution, and neutrophil recovery. As used herein, the term
"T-cell compartment" refers to the cell compartment in a subject
that contains all immature, mature, undifferentiated and
differentiated B-cells. The "T-cell compartment" includes, without
limitation, T-cell progenitors, pro-T-cells, pre-T-cells, double
positive T-cells, immature T-cells, mature T-cells, helper T-cells,
cytotoxic T-cells, memory T-cells, regulatory T-cells, natural
killer T-cells (NKT-cells), and gamma delta T-cells. As used
herein, the term "B-cell compartment" refers to the cell
compartment in a subject that contains all immature, mature,
undifferentiated and differentiated B-cells. The "B-cell
compartment" includes, without limitation, B-cell progenitors,
pro-B-cells, early pro-B-cells, late-pro B-cells, large
pre-B-cells, small pre-B-cells, immature B-cells, mature B-cells,
plasma B-cells, memory B-cells, B-1 cells, B-2 cells, marginal-zone
B-cells, and follicular B-cells. As used herein, the term "NK cell
compartment" refers to the cell compartment in a subject that
contains all progenitor, immature, mature, undifferentiated and
differentiated natural killer (NK) cells. The "NK cell compartment"
NK cells. As used herein, the term "myeloid cell compartment"
refers to the cell compartment in a subject that contains all
progenitor, immature, mature, undifferentiated and differentiated
myeloid cell populations. The "myeloid cell compartment" includes,
without limitation, monocytes, macrophages, neutrophils, basophils,
eosinophils, erythrocytes, megakaryocytes, platelets, and dendritic
cells.
[0246] Accordingly, in certain embodiments, the accelerated
hematopoietic compartment reconstitution in an HSC transplant
recipient that has been administered HSCs pre-treated with a
MYC-composition of the present disclosure, a Bcl-2-composition of
the present disclosure, or both; or administered a MYC-composition
of the present disclosure, a Bcl-2-composition of the present
disclosure, or both results in T-cell compartment reconstitution
that is accelerated by at least about or about 25% to art least
about or about 500%, for example, at least about or about 25%, at
least about or about 30%, at least about or about 31%, at least
about or about 32%, at least about or about 33%, at least about or
about 34%, at least about or about 35%, at least about or about
40%, at least about or about 45%, at least about or about 50%, at
least about or about 55%, at least about or about 60%, at least
about or about 65%, at least about or about 66%, at least about or
about 67%, at least about or about 68%, at least about or about
69%, at least about or about 70%, at least about or about 75%, at
least about or about 80%, at least about or about 90%, at least
about or about 95%, at least about or about 100%, at least about or
about 150%, at least about or about 200%, at least about or about
250%, at least about or about 300%, at least about or about 400%,
at least about or about 500%, or a higher percentage more, compared
to T-cell compartment reconstitution in a subject that is not
administered the pre-treated HSCs; or the MYC-composition, the
Bcl-2-composition, or both.
[0247] In other embodiments, the accelerated hematopoietic
compartment reconstitution in an HSC transplant recipient that has
been administered HSCs pre-treated with a MYC-composition of the
present disclosure, a Bcl-2-composition of the present disclosure,
or both; or administered a MYC-composition of the present
disclosure, a Bcl-2-composition of the present disclosure, or both
results in NKT-cell reconstitution that is accelerated by at least
about or about 25% to art least about or about 500%, for example,
at least about or about 25%, at least about or about 30%, at least
about or about 31%, at least about or about 32%, at least about or
about 33%, at least about or about 34%, at least about or about
35%, at least about or about 40%, at least about or about 45%, at
least about or about 50%, at least about or about 55%, at least
about or about 60%, at least about or about 65%, at least about or
about 66%, at least about or about 67%, at least about or about
68%, at least about or about 69%, at least about or about 70%, at
least about or about 75%, at least about or about 80%, at least
about or about 90%, at least about or about 95%, at least about or
about 100%, at least about or about 150%, at least about or about
200%, at least about or about 250%, at least about or about 300%,
at least about or about 400%, at least about or about 500%, or a
higher percentage more, compared to NKT-cell reconstitution in a
subject that is not administered the pre-treated HSCs; or the
MYC-composition, the Bcl-2-composition, or both.
[0248] In other embodiments, the accelerated hematopoietic
compartment reconstitution in an HSC transplant recipient that has
been administered HSCs pre-treated with a MYC-composition of the
present disclosure, a Bcl-2-composition of the present disclosure,
or both; or administered a MYC-composition of the present
disclosure, a Bcl-2-composition of the present disclosure, or both
results in B-cell compartment reconstitution that is accelerated by
at least about or about 25% to art least about or about 500%, for
example, at least about or about 25%, at least about or about 30%,
at least about or about 31%, at least about or about 32%, at least
about or about 33%, at least about or about 34%, at least about or
about 35%, at least about or about 40%, at least about or about
45%, at least about or about 50%, at least about or about 55%, at
least about or about 60%, at least about or about 65%, at least
about or about 66%, at least about or about 67%, at least about or
about 68%, at least about or about 69%, at least about or about
70%, at least about or about 75%, at least about or about 80%, at
least about or about 90%, at least about or about 95%, at least
about or about 100%, at least about or about 150%, at least about
or about 200%, at least about or about 250%, at least about or
about 300%, at least about or about 400%, at least about or about
500%, or a higher percentage more, compared to B-cell compartment
reconstitution in a subject that is not administered the
pre-treated HSCs; or the MYC-composition, the Bcl-2-composition, or
both.
[0249] In other embodiments, the accelerated hematopoietic
compartment reconstitution in an HSC transplant recipient that has
been administered HSCs pre-treated with a MYC-composition of the
present disclosure, a Bcl-2-composition of the present disclosure,
or both; or administered a MYC-composition of the present
disclosure, a Bcl-2-composition of the present disclosure, or both
results in NK-cell compartment reconstitution that is accelerated
by at least about or about 25% to art least about or about 500%,
for example, at least about or about 25%, at least about or about
30%, at least about or about 31%, at least about or about 32%, at
least about or about 33%, at least about or about 34%, at least
about or about 35%, at least about or about 40%, at least about or
about 45%, at least about or about 50%, at least about or about
55%, at least about or about 60%, at least about or about 65%, at
least about or about 66%, at least about or about 67%, at least
about or about 68%, at least about or about 69%, at least about or
about 70%, at least about or about 75%, at least about or about
80%, at least about or about 90%, at least about or about 95%, at
least about or about 100%, at least about or about 150%, at least
about or about 200%, at least about or about 250%, at least about
or about 300%, at least about or about 400%, at least about or
about 500%, or a higher percentage more, compared to NK-cell
compartment reconstitution in a subject that is not administered
the pre-treated HSCs; or the MYC-composition, the
Bcl-2-composition, or both.
[0250] In other embodiments, the accelerated hematopoietic
compartment reconstitution in an HSC transplant recipient that has
been administered HSCs pre-treated with a MYC-composition of the
present disclosure, a Bcl-2-composition of the present disclosure,
or both; or administered a MYC-composition of the present
disclosure, a Bcl-2-composition of the present disclosure, or both
results in myeloid cell compartment reconstitution that is
accelerated by at least about or about 25% to art least about or
about 500%, for example, at least about or about 25%, at least
about or about 30%, at least about or about 31%, at least about or
about 32%, at least about or about 33%, at least about or about
34%, at least about or about 35%, at least about or about 40%, at
least about or about 45%, at least about or about 50%, at least
about or about 55%, at least about or about 60%, at least about or
about 65%, at least about or about 66%, at least about or about
67%, at least about or about 68%, at least about or about 69%, at
least about or about 70%, at least about or about 75%, at least
about or about 80%, at least about or about 90%, at least about or
about 95%, at least about or about 100%, at least about or about
150%, at least about or about 200%, at least about or about 250%,
at least about or about 300%, at least about or about 400%, at
least about or about 500%, or a higher percentage more, compared to
myeloid cell compartment reconstitution in a subject that is not
administered the pre-treated HSCs; or the MYC-composition, the
Bcl-2-composition, or both.
[0251] In other embodiments, the accelerated hematopoietic
compartment reconstitution in an HSC transplant recipient that has
been administered HSCs pre-treated with a MYC-composition of the
present disclosure, a Bcl-2-composition of the present disclosure,
or both; or administered a MYC-composition of the present
disclosure, a Bcl-2-composition of the present disclosure, or both
results in monocyte reconstitution that is accelerated by at least
about or about 25% to art least about or about 500%, for example,
at least about or about 25%, at least about or about 30%, at least
about or about 31%, at least about or about 32%, at least about or
about 33%, at least about or about 34%, at least about or about
35%, at least about or about 40%, at least about or about 45%, at
least about or about 50%, at least about or about 55%, at least
about or about 60%, at least about or about 65%, at least about or
about 66%, at least about or about 67%, at least about or about
68%, at least about or about 69%, at least about or about 70%, at
least about or about 75%, at least about or about 80%, at least
about or about 90%, at least about or about 95%, at least about or
about 100%, at least about or about 150%, at least about or about
200%, at least about or about 250%, at least about or about 300%,
at least about or about 400%, at least about or about 500%, or a
higher percentage more, compared to monocyte reconstitution in a
subject that is not administered the pre-treated HSCs; or the
MYC-composition, the Bcl-2-composition, or both.
[0252] In other embodiments, the accelerated hematopoietic
compartment reconstitution in an HSC transplant recipient that has
been administered HSCs pre-treated with a MYC-composition of the
present disclosure, a Bcl-2-composition of the present disclosure,
or both; or administered a MYC-composition of the present
disclosure, a Bcl-2-composition of the present disclosure, or both
results in macrophage reconstitution that is accelerated by at
least about or about 25% to art least about or about 500%, for
example, at least about or about 25%, at least about or about 30%,
at least about or about 31%, at least about or about 32%, at least
about or about 33%, at least about or about 34%, at least about or
about 35%, at least about or about 40%, at least about or about
45%, at least about or about 50%, at least about or about 55%, at
least about or about 60%, at least about or about 65%, at least
about or about 66%, at least about or about 67%, at least about or
about 68%, at least about or about 69%, at least about or about
70%, at least about or about 75%, at least about or about 80%, at
least about or about 90%, at least about or about 95%, at least
about or about 100%, at least about or about 150%, at least about
or about 200%, at least about or about 250%, at least about or
about 300%, at least about or about 400%, at least about or about
500%, or a higher percentage more, compared to macrophage
reconstitution in a subject that is not administered the
pre-treated HSCs; or the MYC-composition, the Bcl-2-composition, or
both.
[0253] In other embodiments, the accelerated hematopoietic
compartment reconstitution in an HSC transplant recipient that has
been administered HSCs pre-treated with a MYC-composition of the
present disclosure, a Bcl-2-composition of the present disclosure,
or both; or administered a MYC-composition of the present
disclosure, a Bcl-2-composition of the present disclosure, or both
results in basophil reconstitution that is accelerated by at least
about or about 25% to art least about or about 500%, for example,
at least about or about 25%, at least about or about 30%, at least
about or about 31%, at least about or about 32%, at least about or
about 33%, at least about or about 34%, at least about or about
35%, at least about or about 40%, at least about or about 45%, at
least about or about 50%, at least about or about 55%, at least
about or about 60%, at least about or about 65%, at least about or
about 66%, at least about or about 67%, at least about or about
68%, at least about or about 69%, at least about or about 70%, at
least about or about 75%, at least about or about 80%, at least
about or about 90%, at least about or about 95%, at least about or
about 100%, at least about or about 150%, at least about or about
200%, at least about or about 250%, at least about or about 300%,
at least about or about 400%, at least about or about 500%, or a
higher percentage more, compared to basophil reconstitution in a
subject that is not administered the pre-treated HSCs; or the
MYC-composition, the Bcl-2-composition, or both.
[0254] In other embodiments, the accelerated hematopoietic
compartment reconstitution in an HSC transplant recipient that has
been administered HSCs pre-treated with a MYC-composition of the
present disclosure, a Bcl-2-composition of the present disclosure,
or both; or administered a MYC-composition of the present
disclosure, a Bcl-2-composition of the present disclosure, or both
results in eosinophil reconstitution that is accelerated by at
least about or about 25% to art least about or about 500%, for
example, at least about or about 25%, at least about or about 30%,
at least about or about 31%, at least about or about 32%, at least
about or about 33%, at least about or about 34%, at least about or
about 35%, at least about or about 40%, at least about or about
45%, at least about or about 50%, at least about or about 55%, at
least about or about 60%, at least about or about 65%, at least
about or about 66%, at least about or about 67%, at least about or
about 68%, at least about or about 69%, at least about or about
70%, at least about or about 75%, at least about or about 80%, at
least about or about 90%, at least about or about 95%, at least
about or about 100%, at least about or about 150%, at least about
or about 200%, at least about or about 250%, at least about or
about 300%, at least about or about 400%, at least about or about
500%, or a higher percentage more, compared to eosinophil
reconstitution in a subject that is not administered the
pre-treated HSCs; or the MYC-composition, the Bcl-2-composition, or
both.
[0255] In other embodiments, the accelerated hematopoietic
compartment reconstitution in an HSC transplant recipient that has
been administered HSCs pre-treated with a MYC-composition of the
present disclosure, a Bcl-2-composition of the present disclosure,
or both; or administered a MYC-composition of the present
disclosure, a Bcl-2-composition of the present disclosure, or both
results in erythrocyte reconstitution that is accelerated by at
least about or about 25% to art least about or about 500%, for
example, at least about or about 25%, at least about or about 30%,
at least about or about 31%, at least about or about 32%, at least
about or about 33%, at least about or about 34%, at least about or
about 35%, at least about or about 40%, at least about or about
45%, at least about or about 50%, at least about or about 55%, at
least about or about 60%, at least about or about 65%, at least
about or about 66%, at least about or about 67%, at least about or
about 68%, at least about or about 69%, at least about or about
70%, at least about or about 75%, at least about or about 80%, at
least about or about 90%, at least about or about 95%, at least
about or about 100%, at least about or about 150%, at least about
or about 200%, at least about or about 250%, at least about or
about 300%, at least about or about 400%, at least about or about
500%, or a higher percentage more, compared to erythrocyte
reconstitution in a subject that is not administered the
pre-treated HSCs; or the MYC-composition, the Bcl-2-composition, or
both.
[0256] In other embodiments, the accelerated hematopoietic
compartment reconstitution in an HSC transplant recipient that has
been administered HSCs pre-treated with a MYC-composition of the
present disclosure, a Bcl-2-composition of the present disclosure,
or both; or administered a MYC-composition of the present
disclosure, a Bcl-2-composition of the present disclosure, or both
results in megakaryocyte reconstitution that is accelerated by at
least about or about 25% to art least about or about 500%, for
example, at least about or about 25%, at least about or about 30%,
at least about or about 31%, at least about or about 32%, at least
about or about 33%, at least about or about 34%, at least about or
about 35%, at least about or about 40%, at least about or about
45%, at least about or about 50%, at least about or about 55%, at
least about or about 60%, at least about or about 65%, at least
about or about 66%, at least about or about 67%, at least about or
about 68%, at least about or about 69%, at least about or about
70%, at least about or about 75%, at least about or about 80%, at
least about or about 90%, at least about or about 95%, at least
about or about 100%, at least about or about 150%, at least about
or about 200%, at least about or about 250%, at least about or
about 300%, at least about or about 400%, at least about or about
500%, or a higher percentage more, compared to megakaryocyte
reconstitution in a subject that is not administered the
pre-treated HSCs; or the MYC-composition, the Bcl-2-composition, or
both.
[0257] In other embodiments, the accelerated hematopoietic
compartment reconstitution in an HSC transplant recipient that has
been administered HSCs pre-treated with a MYC-composition of the
present disclosure, a Bcl-2-composition of the present disclosure,
or both; or administered a MYC-composition of the present
disclosure, a Bcl-2-composition of the present disclosure, or both
results in platelet reconstitution that is accelerated by at least
about or about 25% to art least about or about 500%, for example,
at least about or about 25%, at least about or about 30%, at least
about or about 31%, at least about or about 32%, at least about or
about 33%, at least about or about 34%, at least about or about
35%, at least about or about 40%, at least about or about 45%, at
least about or about 50%, at least about or about 55%, at least
about or about 60%, at least about or about 65%, at least about or
about 66%, at least about or about 67%, at least about or about
68%, at least about or about 69%, at least about or about 70%, at
least about or about 75%, at least about or about 80%, at least
about or about 90%, at least about or about 95%, at least about or
about 100%, at least about or about 150%, at least about or about
200%, at least about or about 250%, at least about or about 300%,
at least about or about 400%, at least about or about 500%, or a
higher percentage more, compared to platelet reconstitution in a
subject that is not administered the pre-treated HSCs; or the
MYC-composition, the Bcl-2-composition, or both.
[0258] In other embodiments, the accelerated hematopoietic
compartment reconstitution in an HSC transplant recipient that has
been administered HSCs pre-treated with a MYC-composition of the
present disclosure, a Bcl-2-composition of the present disclosure,
or both; or administered a MYC-composition of the present
disclosure, a Bcl-2-composition of the present disclosure, or both
results in dendritic cell reconstitution that is accelerated by at
least about or about 25% to art least about or about 500%, for
example, at least about or about 25%, at least about or about 30%,
at least about or about 31%, at least about or about 32%, at least
about or about 33%, at least about or about 34%, at least about or
about 35%, at least about or about 40%, at least about or about
45%, at least about or about 50%, at least about or about 55%, at
least about or about 60%, at least about or about 65%, at least
about or about 66%, at least about or about 67%, at least about or
about 68%, at least about or about 69%, at least about or about
70%, at least about or about 75%, at least about or about 80%, at
least about or about 90%, at least about or about 95%, at least
about or about 100%, at least about or about 150%, at least about
or about 200%, at least about or about 250%, at least about or
about 300%, at least about or about 400%, at least about or about
500%, or a higher percentage more, compared to dendritic cell
reconstitution in a subject that is not administered the
pre-treated HSCs; or the MYC-composition, the Bcl-2-composition, or
both.
[0259] In other embodiments, the accelerated hematopoietic
compartment reconstitution in an HSC transplant recipient that has
been administered HSCs pre-treated with a MYC-composition of the
present disclosure, a Bcl-2-composition of the present disclosure,
or both; or administered a MYC-composition of the present
disclosure, a Bcl-2-composition of the present disclosure, or both
results in neutrophil recovery that is accelerated by at least
about or about 25% to art least about or about 500%, for example,
at least about or about 25%, at least about or about 30%, at least
about or about 31%, at least about or about 32%, at least about or
about 33%, at least about or about 34%, at least about or about
35%, at least about or about 40%, at least about or about 45%, at
least about or about 50%, at least about or about 55%, at least
about or about 60%, at least about or about 65%, at least about or
about 66%, at least about or about 67%, at least about or about
68%, at least about or about 69%, at least about or about 70%, at
least about or about 75%, at least about or about 80%, at least
about or about 90%, at least about or about 95%, at least about or
about 100%, at least about or about 150%, at least about or about
200%, at least about or about 250%, at least about or about 300%,
at least about or about 400%, at least about or about 500%, or a
higher percentage more, compared to neutrophil recovery in a
subject that is not administered the pre-treated HSCs; or the
MYC-composition, the Bcl-2-composition, or both.
[0260] Enhanced Hematopoietic Compartment Autoreconstitution
[0261] In other embodiments, administering a MYC-composition of the
present disclosure to a subject in need of autoreconstitution
enhances hematopoietic compartment autoreconstitution in the
subject. For example, administering the MYC-composition may improve
the rate of autoreconstitution, increase the amount of
hematopoietic compartment cells that are formed, or reduce loss of
hematopoietic compartment cells in the subject, as compared to a
subject that is not administered the MYC-composition.
[0262] Administration of a MYC-composition of the present
disclosure, a Bcl-2-composition of the present disclosure, or both
may enhance hematopoietic compartment autoreconstitution by at
least about or about 25% to art least about or about 500%, for
example, at least about or about 25%, at least about or about 30%,
at least about or about 31%, at least about or about 32%, at least
about or about 33%, at least about or about 34%, at least about or
about 35%, at least about or about 40%, at least about or about
45%, at least about or about 50%, at least about or about 55%, at
least about or about 60%, at least about or about 65%, at least
about or about 66%, at least about or about 67%, at least about or
about 68%, at least about or about 69%, at least about or about
70%, at least about or about 75%, at least about or about 80%, at
least about or about 90%, at least about or about 95%, at least
about or about 100%, at least about or about 150%, at least about
or about 200%, at least about or about 250%, at least about or
about 300%, at least about or about 400%, at least about or about
500%, or more, compared to hematopoietic compartment
autoreconstitution in a subject that is not administered the
MYC-composition composition, the Bcl-2-composition, or both.
[0263] As disclosed herein, hematopoietic compartment
autoreconstitution includes, without limitation, T-cell compartment
autoreconstitution, B-cell compartment autoreconstitution, NKT-cell
autoreconstitution, and NK-cell compartment autoreconstitution;
myeloid cell compartment autoreconstitution, such as monocyte
autoreconstitution, macrophage autoreconstitution, basophil
autoreconstitution, eosinophil autoreconstitution, erythrocyte
autoreconstitution, megakaryocyte autoreconstitution, platelet
autoreconstitution, and dendritic cell autoreconstitution; and
neutrophil recovery.
Composition Formulations
[0264] Certain aspects of the present disclosure relate to
compositions containing a MYC-composition of the present
disclosure, a Bcl-2-composition of the present disclosure, or both
for pre-treating HSCs of the present disclosure, and for treating
subjects in need of an HSC transplant. Other aspects relate to a
first composition containing HSCs for achieving hematopoietic
compartment reconstitution in a subject in need thereof, and a
second composition containing a MYC-composition of the present
disclosure, a Bcl-2-composition of the present disclosure, or both
for enhancing hematopoietic compartment reconstitution in the
subject. Other aspects of the present disclosure relate to a
composition containing a MYC-composition of the present disclosure,
a Bcl-2-composition of the present disclosure, or both for
enhancing hematopoietic compartment autoreconstitution in a subject
in need thereof.
[0265] In some embodiments, compositions of the present disclosure
are formulated in a conventional manner using one or more
physiologically acceptable carriers including, e.g., excipients and
auxiliaries which facilitate processing of the active compounds
into preparations which are suitable for pharmaceutical use.
Suitable pharmaceutically acceptable carriers include, without
limitation, saline, aqueous buffer solutions, solvents and/or
dispersion media. The use of such carriers is well known in the
art. The carrier is preferably sterile. In some embodiments, the
carrier is stable under the conditions of manufacture and storage
and preserved against the contaminating action of microorganisms,
such as bacteria and fungi, through the use of, for example and
without limitation, parabens, chlorobutanol, phenol, ascorbic acid,
or thimerosal.
[0266] Examples of materials and solutions that can serve as
pharmaceutically acceptable carriers include, without limitation:
(1) sugars, such as lactose, glucose and sucrose; (2) starches,
such as corn starch and potato starch; (3) cellulose, and its
derivatives, such as sodium carboxymethyl cellulose, ethyl
cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt;
(6) gelatin; (7) talc; (8) excipients, such as cocoa butter and
suppository waxes; (9) oils, such as peanut oil, cottonseed oil,
safflower oil, sesame oil, olive oil, corn oil and soybean oil;
(10) glycols, such as propylene glycol; (11) polyols, such as
glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,
such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering
agents, such as magnesium hydroxide and aluminum hydroxide; (15)
alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)
Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions;
(21) polyesters, polycarbonates and/or polyanhydrides; and (22)
other non-toxic compatible substances employed in pharmaceutical
formulations.
[0267] Proper formulation of the compositions of the present
disclosure may be dependent upon the route of administration
chosen. A summary of pharmaceutical compositions described herein
is found, for example, in Remington: The Science and Practice of
Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company,
1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack
Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L.,
Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.,
1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems,
Seventh Ed. (Lippincott Williams & Wilkins 1999).
[0268] The compositions of the present disclosure can facilitate
administration of a compound of the present disclosure (e.g., HSCs
or a MYC-composition or a Bcl-2-composition) to a subject or cell.
In certain embodiments of the methods of the present disclosure,
therapeutically effective amounts of compounds described herein are
administered in a pharmaceutical composition to a subject having a
disorder, disease, or condition to be treated. In some embodiments,
the subject is a human patient. In other embodiments, the subject
is a non-human animal, including without limitation, a dog, cat,
horse, cow, pig, sheep, goat, chicken, monkey, rat, mouse, or the
like.
[0269] The HSC compositions, MYC-composition-containing
compositions, and/or Bcl-2-composition-containing compositions of
the present disclosure may be utilized either alone or in
combination with one or more additional therapeutic agents. For
example, cytokines, growth factors, antibodies, and/or small
molecule modifiers that are known to aid in reducing the time
required to reconstitute mature hematopoietic lineages after HSC
transplantation, may administered in combination with the HSC,
MYC-composition-containing, and/or Bcl-2-composition-containing
compositions. For example, monoclonal antibodies and/or small
molecule modifiers that target enzymes affecting E-selectin
expression can improve HSC homing to bone marrow niches after an
HSC transplantation. Other examples include, without limitation,
antibodies or small molecules that affect adhesion proteins such
and 136 integrin, G-MCSF, G-CSF is the same, and Epo. Accordingly,
certain embodiments of the methods of the present disclosure
further include administering a third composition containing at
least one cytokine, growth factor, antibody, and/or small molecule
modifier. In some embodiments, the cytokine and/or growth factor
composition further contains a pharmaceutically acceptable
carrier.
[0270] The compositions of the present disclosure may be
administered to a subject in any suitable manner, including,
without limitation, one or more of multiple administration routes,
such as, oral, parenteral (e.g., intravenous, subcutaneous,
intramuscular), intranasal, buccal, rectal, or transdermal
administration routes.
[0271] Composition formulations of the present disclosure include,
without limitation, aqueous liquid dispersions, oil emulsions,
self-emulsifying dispersions, solid solutions, liposomal
dispersions, aerosols, solid dosage forms, powders, immediate
release formulations, controlled release formulations, fast melt
formulations, tablets, capsules, pills, delayed release
formulations, extended release formulations, pulsatile release
formulations, multiparticulate formulations, and mixed immediate
and controlled release formulations.
[0272] Pharmaceutical compositions of the present disclosure (e.g.,
a PTD-MYC composition, a PTD-Bcl-2 composition, or a cytokine
and/or growth factor composition) are optionally manufactured in a
conventional manner, including, without limitation, conventional
mixing, dissolving, granulating, dragee-making, levigating,
emulsifying, encapsulating, entrapping or compression
processes.
[0273] In some embodiments, pharmaceutical compositions of the
present disclosure (e.g., a PTD-MYC composition, a PTD-Bcl-2
composition, or a cytokine and/or growth factor composition) are in
unit dosage forms suitable for single administration of precise
dosages. In unit dosage form, the formulation is divided into unit
doses containing appropriate quantities of one or more compound. In
some embodiments, the unit dosage is in the form of a package
containing discrete quantities of the formulation. Non-limiting
examples include packaged tablets or capsules, and powders in vials
or ampoules. Aqueous suspension compositions are optionally
packaged in single-dose non-reclosable containers. In some
embodiments, multiple-dose re-closeable containers are used. In
certain embodiments, multiple dose containers contain a
preservative in the composition. Formulations for parenteral
injection may be presented in unit dosage forms, which include,
without limitation, ampoules or in multi-dose containers with an
added preservative.
[0274] The following Examples are merely illustrative and are not
meant to limit any aspects of the present disclosure in any
way.
EXAMPLES
Example 1: Accelerated Hematopoietic Reconstitution in Mice
[0275] The following example describes the results of treating mice
with a TAT-MYC fusion protein after transplantation with expanded
bone marrow cells (e.g. protein transduced longterm hematopoietic
stem cells (ptlt-HSCs)).
[0276] Materials and Methods
[0277] Cohorts of 4-6 week old female C57BL/6J mice were obtained
from Jackson Laboratories (Bar Harbor, Me.). The mice were treated
with 5 mg/mouse of 5-fluorouracil (5FU), intravenously. Bone marrow
(BM) cells were collected from the tibia and femur bones 5 days
after 5FU treatment. The red blood cells were lysed by incubation
in 5 ml sterile TAC buffer (135 mM NH4CL, 17 mM Tris Ph 7.65). The
bone marrow cells were expanded in BM Medium (DMEM containing 15%
FCS, 100 units per ml Penn/Strep, MEM NEAA (Gibco), 10 mM HEPES,
recombinant murine IL-3, IL-6, and SCF) supplemented with 5
.mu.g/ml recombinant Tat-Myc, and 10 .mu.g/ml recombinant
Tat-Bcl-2. Cells were cultured for 21 days with a BM medium change
every 48 hr to refresh the Tat-fusion proteins.
[0278] Cytokines were prepared by plating 293FT cells in 150 mm
plates at 12.times.10.sup.6 cells per plate in D10 media (DMEM, 10%
FBS, 100 units per ml Penn/Strep, MEM NEAA (Gibco), 2 mM
L-glutamine (Gibco)). The cells were transfected with 30 .mu.g
total DNA per plate consisting of 10 .mu.g pcDNA3.1-SCF, 10 .mu.g
pcDNA3.1-IL3, and 10 .mu.g pcDNA3.1-IL6 or 10 .mu.g pcDNA3.1-TPO,
10 .mu.g pcDNA3.1-Flt3-L, and 10 .mu.g pcDNA3.1-GM-CSF using
calcium phosphate (Young, R. M., et al. (2008). Future Oncology, 4,
591-4.). The following day, the media was removed and was replaced
with 100 ml D10 media. Cells were incubated at 37.degree. C./5% CO2
for 4-5 days. The media was collected, sterile filtered, and frozen
at -20.degree. C. in 30 ml aliquots.
[0279] After 21 days, 5.times.10.sup.3 expanded bone marrow cells
were transplanted into sublethally irradiated Rag-1.sup.-/- mice on
a C57/BL6 background (Jackson Laboratory) that received 350 Rads of
radiation just prior to injection of the BM cells via the tail
vein. The expanded cells were washed 3 times in PBS prior to
injection via the tail vein in 200 .mu.l PBS.
[0280] After 24 hours, one cohort of mice was injected
intramuscularly with 10 .mu.g TAT-MYC emulsified in corn oil. The
emulsion is prepared by adding 30 .mu.g Tat-MYC into 1 ml of corn
oil. Just prior to injection, the corn oil containing the Tat-MYC
is passed several times from 1 syringe to a second syringe through
a 2-way stopcock. 300 ul of the emulsion containing 10 .mu.g of
Tat-MYC is injected IM into the mouse, just to the side of the
tail.
[0281] Reconstitution of the lymphoid compartment was monitored by
flow cytometric analysis (FACS) of peripheral blood samples
obtained by venipuncture of the tail at 4 weeks and 8 weeks
following transplant. Peripheral blood mononuclear cells (PBMCs)
were monitored by FACS for TCR.beta. and for B220 expression.
[0282] Results
[0283] As shown in FIGS. 1 and 3, accelerated development of
T-cells was seen in mice treated with TAT-MYC following bone marrow
transplant with ex vivo expanded bone marrow cells. Cohorts of
Rag-1.sup.-/- mice were sub-lethally irradiated and given
transplants of 5.times.10.sup.3 expanded bone marrow cells (FIGS.
1C and 1D; FIGS. 3C and 3D). Half of the irradiated and
transplanted mice in each cohort were injected with 10 .mu.g
TAT-MYC 24 hours after the transplant (FIGS. 1D and 3D). FACS
analysis of wild-type (untreated and non-irradiated) Rag-1.sup.-/-
mice (FIGS. 1A, 0.9% TCR.beta.+ cells and 3A, 0.8% TCR.beta..sup.+
cells) and C57BL/6 mice (FIG. 1B, 34.3% TCR.beta..sup.+ cells and
3B, 34.3% TCR.beta..sup.+ cells) are also provided as a
control.
[0284] Mice were tested for T cell reconstitution at 4 weeks (FIG.
1) and 8 weeks (FIG. 3) by FACS analysis of their peripheral blood.
FIG. 1D shows the 4-week levels of T cells in the peripheral blood
of mice treated with TAT-MYC (5.6% TCR.beta..sup.+ cells), as
compared to the control (FIG. 1C) that was not injected with
TAT-MYC (0.2% TCR.beta..sup.+ cells). FIG. 3D shows the 8-week
levels of T cells in the peripheral blood of mice treated with
TAT-MYC (13.1% TCR.beta..sup.+ cells), as compared to the control
(FIG. 3C) that was not injected with TAT-MYC (4.2% TCR.beta..sup.+
cells). At 4 weeks, the TAT-MYC injected mice showed T cell levels
similar to those of control mice not injected with TAT-MYC at 8
weeks. As the average time to T-cell recovery is 8-10 weeks, this
represents an acceleration of about 50-60%.
[0285] As shown in FIGS. 2 and 4, accelerated development of B
cells was also seen in mice treated with TAT-MYC following bone
marrow transplant with expanded bone marrow cells.
[0286] Cohorts of Rag-1.sup.-/- mice on a C57/BL6 background were
sub-lethally irradiated and given transplants of 5.times.10.sup.3
expanded bone marrow cells. Half of the irradiated and transplanted
mice in each cohort were injected with 10 .mu.g TAT-MYC 24 hours
after the transplant (FIGS. 2D and 4D). FACS analysis of wild-type
(untreated and non-irradiated) Rag-1.sup.-/- mice (FIG. 2A, 1.15%
B220+ cells and 4A, 1% B220+ cells) and C57BL/6 mice are also
provided as a control (FIGS. 2B, 21.4% B220+ cells and 4B, 29.2%
B220+ cells).
[0287] Mice were tested for B cell reconstitution at 4 weeks (FIG.
2) and 8 weeks (FIG. 4) by FACS analysis of their peripheral blood.
FIG. 2D shows the 4-week levels of B cells in the peripheral blood
of mice treated with TAT-MYC (12.1% B220+ cells), as compared to
the control (FIG. 2C) that was not injected with TAT-MYC (0.3%
B220+ cells). FIG. 4D shows the 8-week levels of B cells in the
peripheral blood of mice treated with TAT-MYC (5.4% B220+ cells),
as compared to the control (FIG. 4C) that was not injected with
TAT-MYC (1.2% B220+ cells). At 4 weeks, the TAT-MYC injected mice
showed B cell levels higher than those of control mice not injected
with TAT-MYC at 8 weeks. As the average time to B-cell recovery is
8-12 weeks, this represents an acceleration of about 50-67%.
Example 2: Accelerated Hematopoietic Reconstitution in Mice
[0288] The following example describes the results of treating mice
with a TAT-MYC fusion protein following freshly isolated whole bone
marrow transplantation.
[0289] Materials and Methods
[0290] For whole bone marrow transplantations in mice, donors and
recipients were both on a C57/BL6 background.
[0291] Bone marrow cells were flushed from femurs and tibial bones
obtained from two donor wild type C57/BL6 mice. The harvested cells
were transferred into D10 complete medium (DMEM supplemented with
10% heat inactivated fetal calf serum, 100 units/ml
penicillin/streptomycin, 10 .mu.g/ml L-glutamine, as well as MEM
NEAA). The bone marrow aspirates were dissociated into single cell
suspensions and pelleted by centrifugation. The red blood cells
were lysed by incubation of the cell suspension in a hypotonic
buffer (135 mM NH.sub.4Cl, 17 mM Tris, pH 7.65). The remaining
cells were then washed in D10 medium followed by two washes with
PBS and kept cold until transplantation into Rag-1.sup.-/- mice the
same day.
[0292] The recipient Rag-1.sup.-/- mice were irradiated with 350
Rads (whole body irradiation). Recipient mice were given
1.times.10.sup.6 whole bone marrow cells via tail vein injection.
24 hours after the bone marrow cell transplant, mice were given 10
.mu.g of TAT-MYC emulsified in 300 .mu.l of corn oil as described
in Example 1. The TAT-MYC was delivered via intramuscular
injection.
[0293] Reconstitution of the lymphoid compartment was monitored by
flow cytometric analysis (FACS) of peripheral blood samples
obtained by venipuncture of the tail. Specifically, samples were
monitored for the appearance of CD4 or CD8 expressing T-cells
(TCR.beta.), as well as B-cells (CD19 and B220 expressing
cells).
[0294] Spleen cell samples from chimeric mice were also measured
for the ability to respond to mitogenic stimulation. Spleen-derived
T-cells and B-cells were labeled with CFSE and activated with
either antibodies to CD3 (T-cells), or antibodies to IgM and CD40
(B-cells). The cells were evaluated for proliferation, as
determined by dilution of the CFSE signal, using FACS, 72 hours
after stimulation.
[0295] Results
[0296] Cohorts of 5 mice each (Rag-1.sup.-/- mice) were
sub-lethally irradiated and given transplants of 10.sup.6 whole BM
cells obtained from female C57/BL6 donor mice. The transplant
recipient mice were then either injected with TAT-MYC 24 hours
later or not treated. The chimaeric mice were maintained in the
vivarium for observation for 4 weeks. At that point, the mice were
euthanized, and PBMCs and spleens were collected. The spleens were
used to generate single cell suspension. Those cells were then
stained with fluoresceinated antibodies to murine CD4 and CD8, and
analyzed by flow cytometry.
[0297] FIGS. 5 and 6 show the accelerated development of T cells in
mice treated with TAT-MYC following freshly isolated whole bone
marrow transplant. Cohorts of Rag-1.sup.-/- mice on a C57/BL6
background were sub-lethally irradiated and given transplants of
1.times.10.sup.6 whole bone marrow cells. Half of the transplanted
cohorts were injected with TAT-MYC 24 hours after the
transplant.
[0298] FIG. 5A shows the level of peripheral blood CD4 and CD8 T
cells in control Rag-1.sup.-/- mice that did not receive
irradiation, a cell transplant, or treatment with Tat-Myc (0.03%
CD4.sup.+ and 0.8% CD8.sup.+ cells). FIG. 5B shows the level of
peripheral blood CD4 and CD8 T cells in untreated and
non-irradiated wildtype C57BL/6 mice as a control (13.1% CD4.sup.+
and 11.1% CD8.sup.+ cells). FIG. 5D shows the detection of both CD4
and CD8 T-cells in the peripheral blood of mice treated with
TAT-MYC (14.9% CD4.sup.+ and 8.6% CD8 cells), as compared to the
mice (FIG. 5C) that were not injected with TAT-MYC (4.2% CD4.sup.+
and 3.2% CD8 cells). At 4 weeks, mice treated with TAT-MYC 24 hours
after whole bone marrow transplant have approximately the same
levels of CD4 and CD8 T cells as wildtype C57BL/6 mice.
[0299] FIG. 6 graphically depicts the percentage of T cells for the
full cohort of mice described above and shown in FIG. 5. FIG. 6A
shows the percent CD4.sup.+ cells in the peripheral blood at 4
weeks post-transplant in wild type C57BL/6 (First column), in
Rag-1.sup.-/- mice irradiated and transplanted, but not treated
with TAT-MYC (Second column), and in Rag-1.sup.-/- mice irradiated,
transplanted, and injected with 10 .mu.g TAT-MYC (Third column).
FIG. 6B shows the percent CD8.sup.+ cells in the peripheral blood
at 4 weeks post-transplant in wild type C57BL/6 (First column), in
Rag-1.sup.-/- mice irradiated and transplanted, but not treated
with TAT-MYC (Second column), and in Rag-1.sup.-/- mice irradiated,
transplanted, and injected with 10 .mu.g TAT-MYC (Third
column).
[0300] FIGS. 7 and 8 show the accelerated development of B-cells in
mice treated with TAT-MYC following freshly isolated bone marrow
transplant. Cohorts of Rag-1.sup.-/- mice on a C57/BL6 background
were sub-lethally irradiated and given transplants of
1.times.10.sup.6 whole bone marrow cells. Half of the transplanted
cohorts were injected with TAT-MYC 24 hours after the
transplant.
[0301] FIG. 7A shows the level of peripheral blood CD19.times.B220
B cells in control Rag-1.sup.-/- mice that did not receive
irradiation, a cell transplant, or treatment with TAT-MYC (0.2%
CD19.sup.+.times.B220.sup.+ cells). FIG. 7B shows the level of
peripheral blood CD19.times.B220 B cells in untreated and
non-irradiated wildtype C57BL/6 mice as a control (25.8%
CD19.sup.+.times.B220.sup.+ cells). FIG. 7D shows the detection of
both CD19.times.B220 B cells in the peripheral blood of mice
treated with TAT-MYC (5.2% CD19.sup.+.times.B220.sup.+ cells), as
compared to the mice (FIG. 7C) that were not injected with TAT-MYC
(1.4% CD19.sup.+.times.B220.sup.+ cells). At 4 weeks, mice treated
with TAT-MYC 24 hours after whole bone marrow transplant have
significantly higher levels of CD19.times.B220 B cells as
transplanted control mice not treated with TAT-MYC.
[0302] FIG. 8 graphically depicts the percentage of B cells for the
full cohort of mice described above and shown in FIG. 7. FIG. 8A
shows the percent CD19.times.B220.sup.+ cells in the peripheral
blood at 4 weeks post-transplant in wild type C57BL/6 (First
column), in Rag-1.sup.-/- mice irradiated and transplanted, but not
treated with TAT-MYC (Second column), and in Rag-1.sup.-/- mice
irradiated, transplanted, and injected with 10 .mu.g TAT-MYC (Third
column).
[0303] FIG. 9 shows that T-cells and B-cells that developed in
chimeric mice transplanted with whole bone marrow and treated with
TAT-MYC 24 hours after transplant were functional and proliferated
following stimulation of their antigen receptors. Spleen-derived
T-cells and B-cells were labeled with CFSE and activated with
either antibodies to CD3 (T-cells), or antibodies to IgM and CD40
(B-cells). The cells were evaluated for proliferation, as
determined by dilution of the CFSE signal, using FACS, 72 hours
after stimulation.
[0304] As shown in FIG. 9A, 33.3% of the spleen cells from mice
given transplants of 1.times.10.sup.6 whole BM cells, but not
treated with TAT-MYC, showed T cell blasting upon stimulation with
anti-CD3. The spleen cells from the chimeric mouse given
transplants of 1.times.10.sup.6 whole BM cells and treated with
TAT-MYC showed 36.6% T cells blasting after CD3 stimulation (FIG.
9C). Similarly, FIG. 9B shows 6.92% of the spleen cells from mice
given transplants of 1.times.10.sup.6 whole BM cells, but not
treated with TAT-MYC showed B cell blasting upon stimulation with
anti-CD40 and anti-IgM. The spleen cells from the chimeric mouse
given transplants of 1.times.10.sup.6 whole BM cells and treated
with TAT-MYC showed 15.8% B cells blasting after CD40 and IgM
stimulation (FIG. 9D). These data show that the mature lymphoid
cells obtained from HSC chimaeric mice that received TAT-MYC
treatment were able to blast and undergo cell division following
activation through their antigen receptors.
Example 3: Induction of Hematopoietic Reconstitution in Mice
Treated with 5-Fluorouracil
[0305] The following example describes the results of treating mice
with a TAT-MYC fusion protein after administration of
5-fluorouracil (5-FU), a chemotherapeutic agent known to be toxic
to the hematopoietic compartment.
[0306] Introduction
[0307] Current approaches for reversing bone marrow failure that
arises from various environmental insults or disease, among others,
basically rely on the administration of growth factors and red
blood cell transfusions for supportive therapy. The ultimate goal
of these approaches is to encourage the remaining endogenous HSCs
to mobilize and repopulate the hematopoietic compartment
(auto-reconstitution). However, the current approaches are
inefficient and in many cases simply delay the requirement for a
myeloablative bone marrow transplant.
[0308] In addition, the sensitivity of HSCs to chemotherapeutic
drugs as well as radiation has historically limited the doses of
each therapy that can be applied to a patient with a solid tumor,
for example. The loss of HSCs and hematopoietic compartment are one
of the early signs of therapy-related toxicity in cancer patients.
The ability to spare the HSC compartment from the therapeutic
agents used for cancer could significantly change the approaches
that are currently used to deliver such treatments.
[0309] The above results in Examples 1 and 2 utilizing TAT-MYC in
the context of transplantation of HSCs with a low number of donor
cells showed that TAT-MYC could also be useful in promoting
auto-reconstitution of the hematopoietic compartment in patients
with bone marrow failure syndrome. Although not intending to be
bound by theory, it is believed that treatment with TAT-MYC would
target a small number of the remaining resident HSCs and induce
such HSCs to divide and give rise to differentiated hematopoietic
lineages.
[0310] Material and Methods
[0311] Cohorts of 4-6 week old female C57/BL6 wild type (WT) mice
were used for these experiments. Cohorts of 5 mice were treated
intravenously with 5-flouorouracil (5 mg/mouse) alone, or followed
by a treatment with either 10 .mu.g/mouse of TAT-MYC or 10
.mu.g/mouse of TAT-Cre (24 hours post-5FU challenge). Both proteins
were emulsified in corn oil immediately prior to intramuscular
injection. In some experiments, mice were pretreated (48 hours in
advance of 5FU challenge) with either 10 .mu.g/mouse of TAT-MYC or
TAT-Cre.
[0312] Reconstitution of the lymphoid compartment was monitored by
flow cytometric analysis (FACS) of peripheral blood samples
obtained by venipuncture of the tail. Specifically, samples were
monitored for the appearance of CD4 or CD8 expressing T-cells
(TCR.beta.), as well as B-cells (CD19 and B220 expressing
cells).
[0313] Results
[0314] Challenge with 5-flourouracil was used both as a model for
environmental insult, and more broadly as a model for treatment of
any bone marrow failure. FIGS. 10, 11 and 12 show the accelerated
auto-reconstitution of T cells at 2 weeks in mice treated with
TAT-MYC 24 hours following 5-fluorouracil challenge. Cohorts of
C57BL/6 mice were challenged with 5FU and either left untreated
(FIG. 10A), or injected intramuscularly with a control protein
TAT-CRE (FIG. 10B) or with TAT-MYC 24 hours after the 5FU challenge
(FIG. 10C).
[0315] FIG. 10A shows the level of peripheral blood CD4 and CD8
positive T cells at 2 weeks in 5FU challenged C57/BL6 mice that did
not receive treatment with TAT-Cre or TAT-MYC following 5FU
challenge (3.9% CD4.sup.+ and 2.4% CD8.sup.+ cells). FIG. 10B shows
the level of peripheral blood CD4 and CD8 positive T cells in 5FU
challenged C57/BL6 mice that received treatment with 10 .mu.g
TAT-Cre following 5FU challenge (5.5% CD4.sup.+ and 2.55% CD8.sup.+
cells). FIG. 5C shows the level of peripheral blood CD4 and CD8
positive T cells in 5FU challenged C57/BL6 mice that received
treatment with 10 .mu.g TAT-MYC following 5FU challenge (14.9%
CD4.sup.+ and 9.69% CD8.sup.+ cells.
[0316] FIGS. 11 and 12 graphically depict the percentage of T cells
for the full cohort of mice described above and shown in FIG. 10.
FIG. 11 shows the percent CD4.sup.+ cells in the peripheral blood
at 2 weeks post-5FU challenge in C57BL/6 mice not treated with
either protein (First column), in C57BL/6 mice injected with 10
.mu.g TAT-MYC (Second column), and in C57BL/6 mice injected with 10
.mu.g TAT-CRE (Third column). FIG. 12 shows the percent CD8.sup.+
cells in the peripheral blood at 2 weeks post-5FU challenge in
C57BL/6 mice not treated with either protein (First column), in
C57BL/6 mice injected with 10 .mu.g TAT-MYC (Second column), and in
C57BL/6 mice injected with 10 .mu.g TAT-CRE (Third column).
[0317] In other experiments, challenge with 5-flourouracil is used
as a model for protection against environmental insult, including
for protection for HSCs against side-effects of chemotherapy or
radiation therapy.
[0318] Cohorts of C57BL/6 mice are left untreated, treated
intramuscularly with TAT-MYC, or treated intramuscularly with a
control protein (TAT-CRE). Five mg of 5-FU are administered
intravenously 24 hours later. At various times after 5FU challenge
(optionally Days 3, 5, 7 or more), peripheral blood samples are
analyzed by FACS to assess the frequency of T-cells (CD4+ and
CD8.sup.+ T-cells) in the blood. A comparison between mice
pre-treated with TAT-MYC or the control protein TAT-CRE will
indicate whether TAT-MYC is able to confer chemoprotection to
hematopoietic lineages.
Example 4: Induction of Hematopoietic Reconstitution in Mice
Treated with Genotoxic Stress Agents
[0319] In the following example two forms of genotoxic stresses for
HSCs, chemical and radiological, are used. Age and gender matched
C57/BL6 mice are treated with either TAT-MYC or a control protein,
and then subjected to exposure with either a chemotherapeutic agent
such as (5FU) or cyclophosphamide (CTX), or with sub-lethal doses
of radiation. Fluctuations in the frequency of mature T-cell and
B-cells in the peripheral blood of the mice are monitored, starting
5-7 days after exposure to the specific stimuli.
[0320] Use of TAT-MYC to Confer Chemoprotection to Hematopoietic
Lineages In Vivo
[0321] Cohorts of 10 C57/BL6 mice are injected with TAT-MYC,
injected with a negative control (e.g. TAT-CRE), or left untreated.
24, 48, or 72 hours later (optionally any time point between 1 and
72 hours), we will treat 5 mice in each cohort with 5 mg/mouse of
5FU or 4 mg/mouse of cyclophosphamide (CTX). The other 5 mice of
the cohort will be left with only the initial treatment of TAT-MYC,
TAT-CRE, or no injection). Peripheral blood is obtained by
venipuncture, and the frequency of mature T-cells and B-cell cells
in the blood is analyzed by FACS.
[0322] The mice are assessed for reduced changes in the frequency
of murine T-cells and B-cells in the peripheral blood of mice
treated with TAT-MYC prior to challenge with 5FU, Busulfan A or
Cyclophosphamide (CTX), in contrast to the significant decrease in
the frequency of mature T-cells and B-cells observed in all other
mice exposed to those chemotherapeutic agents. Alternatively, the
mice are assessed for deceased recovery time to restore the
frequency of murine T-cells and B-cells in the peripheral blood of
mice treated with TAT-MYC prior to challenge with either 5FU,
Busulfan A or CTX, in compared with the length time needed to see
recovery in the frequency of mature T-cells and B-cells observed in
all other mice exposed to those chemotherapeutic agents.
[0323] Additional studies involve a dose-escalation (or
de-escalation) of either 5FU, Busulfan A or CTX on mice that are
treated with TAT-MYC to determine the parameters of increases in
doses of chemotherapetic agents that are enabled by TAT-MYC
treatment.
[0324] Use of TAT-MYC to Confer Radioprotection to Hematopoietic
Lineages In Vivo
[0325] The experimental setup is essentially the same as above. The
only difference is that the mice are exposed to a range of doses of
radiation rather than to chemotherapeutic agents following
treatment with TAT-MYC, TAT-CRE, or no. Accordingly, 350 Rads and
600 Rads may be used for the C57/BL6J mice. The peripheral blood is
monitored for the fluctuation of mature lymphoid or myeloid cells
in by FACS.
[0326] The mice are assessed for reduced changes in the frequency
of murine T-cells and B-cells in the peripheral blood of mice
treated with TAT-MYC prior to challenge with sub-lethal or lethal
radiation, in contrast to the significant decrease in the frequency
of mature T-cells and B-cells observed in all other mice exposed to
radiation. Alternatively, the mice are assessed for deceased
recovery time to restore the frequency of murine T-cells and
B-cells in the peripheral blood of mice treated with TAT-MYC prior
to challenge with radiation, in compared with the length time
needed to see recovery in the frequency of mature T-cells and
B-cells observed in all other mice exposed to radiation.
[0327] Additional studies involve a dose-escalation (or
de-escalation) of doses of radiation on mice that are treated with
TAT-MYC to determine the parameters of increases in doses of
radiation that are enabled by TAT-MYC treatment, and possible
combination of radiation and chemotherapeutic agents.
Example 5: Accelerated Hematopoietic Reconstitution in Mice
Following Different Routes of Administration
[0328] The following example describes the results of treating mice
with a TAT-MYC fusion protein administered either intramuscularly
or intravenously following freshly isolated whole bone marrow
transplantation.
[0329] Materials and Methods
[0330] Experiments were performed as described in Example 2 accept
that TAT-MYC was provided using different routes of
administration.
[0331] Briefly, mice donors and recipients were both on a C57BL/6
background. Bone marrow cells were flushed from femurs and tibial
bones obtained from two donor wild type C57/BL6 mice. The bone
marrow aspirates were dissociated into single cell suspensions,
pelleted by centrifugation, and the red blood cells lysed. The
remaining cells were then washed and kept cold until
transplantation into Rag-1.sup.-/- mice the same day.
[0332] The recipient Rag-1.sup.-/- mice were irradiated with 350
Rads (whole body irradiation). Recipient mice were given
1.times.10.sup.6 whole bone marrow cells via tail vein injection.
24 hours after the bone marrow cell transplant, mice received an
intravenous injection of 10 .mu.g of TAT-MYC dissolved in 200 .mu.l
PBS or intramuscular injection of 10 .mu.g of TAT-MYC emulsified in
300 .mu.l of corn oil as described in Example 1.
[0333] Reconstitution of the lymphoid compartment was monitored by
flow cytometric analysis (FACS) of peripheral blood samples
obtained by venipuncture of the tail. Specifically, samples were
monitored for the appearance of CD4 or CD8 expressing T-cells
(CD4.times.TCR.beta.), as well as B-cells (IgM.times.CD19
expressing cells).
[0334] Results
[0335] Cohorts of 5 mice each (Rag-1.sup.-/- mice) were
sub-lethally irradiated and given transplants of 10.sup.6 whole
bone marrow cells obtained from female C57/BL6 donor mice. The
transplant recipient mice were then either injected with TAT-MYC 24
hours later or not treated. The chimaeric mice were maintained in
the vivarium for observation for at least 4 weeks, at which point
peripheral blood was obtained by venipuncture and levels of T and B
cells were assessed by FACS.
[0336] FIGS. 13 and 14 show the accelerated development of T cells
in mice treated with TAT-MYC following freshly isolated whole bone
marrow transplant. Cohorts of Rag-1.sup.-/- mice on a C57/BL6
background were sub-lethally irradiated and given transplants of
1.times.10.sup.6 whole bone marrow cells (FIGS. 13C, 13D, and 13E).
Two-thirds of the transplanted mice in the cohort received
intravenous (FIG. 13D) or intramuscular (FIG. 13E) injection with
10 .mu.g TAT-MYC 24 hours after the transplant. FACS analysis of
wild-type (untreated and non-irradiated) Rag-1.sup.-/- mice (FIG.
13A, 0.579% CD4.times.TCR.beta..sup.+ cells) and C57BL/6 mice (FIG.
13B, 17.5% CD4.times.TCR.beta..sup.+ cells) are also provided as a
control.
[0337] FIGS. 13D and 13E show the detection of
CD4.times.TCR.beta..sup.+ T cells in the peripheral blood of mice
receiving intravenous TAT-MYC (32.8% CD4.times.TCR.beta..sup.+
cells) or intramuscular TAT-MYC (18.2% CD4.times.TCR.beta..sup.+
cells), as compared to the control (FIG. 13C) that was not injected
with TAT-MYC (6.39% CD4.times.TCR.beta..sup.+ cells). TAT-MYC
injected by either route resulted in mice having approximately the
same levels of CD4.times.TCR.beta..sup.+ T cells as wildtype
C57BL/6 mice.
[0338] FIG. 14 graphically depicts the percentage of T cells for
the full cohort of mice described above and shown in FIG. 13. The
graph shows the percent CD4.sup.+ cells in the peripheral blood at
4 weeks post-transplant in wild type Rag-1.sup.-/- mice (NT), in
Rag-1.sup.-/- mice irradiated, transplanted, and receiving an
intravenous injection of TAT-MYC (IV), and in Rag-1.sup.-/- mice
irradiated, transplanted, and receiving an intramuscular injection
of TAT-MYC (IM).
[0339] FIGS. 15 and 16 show the accelerated development of B cells
in mice treated with TAT-MYC following freshly isolated whole bone
marrow transplant. Cohorts of Rag-1.sup.-/- mice on a C57/BL6
background were sub-lethally irradiated and given transplants of
1.times.10.sup.6 whole bone marrow cells (FIGS. 15C, 15D and 15E).
Two-thirds of the transplanted mice in the cohort received
intravenous (FIG. 15D) or intramuscular (FIG. 15E) injection with
10 .mu.g TAT-MYC 24 hours after the transplant. FACS analysis of
wild-type (untreated and non-irradiated) Rag-1.sup.-/- mice (FIG.
15A, 0.071% IgM.times.CD19.sup.+ cells) and C57BL/6 mice (FIG. 15B,
23.5% IgM.times.CD19.sup.+ cells) are also provided as a
control.
[0340] FIGS. 15D and 15E show the detection of IgM.times.CD19.sup.+
B cells in the peripheral blood of mice receiving intravenous
TAT-MYC (FIG. 15D, 2.41% IgM.times.CD19.sup.+ cells) or
intramuscular TAT-MYC (FIG. 15E, 6.53% IgM.times.CD19' cells), as
compared to the control (FIG. 15C) that was not injected with
TAT-MYC (1.49% IgM.times.CD19.sup.+ cells). TAT-MYC injected by
either route resulted in mice having higher levels of
IgM.times.CD19.sup.+ B cells than Rag-1.sup.-/- mice transplanted
with whole bone marrow, but not treated with TAT-MYC.
[0341] FIG. 16 graphically depicts the percentage of B cells for
the full cohort of mice described above and shown in FIG. 15. The
graph shows the percent CD19.times.B220.sup.+ cells in the
peripheral blood at 4 weeks post-transplant in wild type
Rag-1.sup.-/- mice (NT), in Rag-1.sup.-/- mice irradiated,
transplanted, and receiving an intravenous injection of TAT-MYC
(IV), and in Rag-1.sup.-/- - mice irradiated, transplanted, and
receiving an intramuscular injection of TAT-MYC (IM).
Example 6: Accelerated Hematopoietic Reconstitution in Mice
Transplanted with Human Cord Blood-Derived HSCs Expanded with
TAT-NYC and TAT-Bcl-2
[0342] The following example describes the results of expanding
cord blood derived HSCs in vitro using TAT-MYC and TAT-Bcl-2 to
form protein transduced longterm hematopoietic stem cells
(ptlt-HSCs), prior to transplantation into sublethally irradiated
mice.
[0343] Material and Methods
[0344] Fresh cord blood cells were obtained from samples that were
discarded from a local cord blood bank. All human cells were
de-identified and exempt from IRB oversight. The total cord volume
was split into 20 ml aliquots and diluted 1:1 in PBS. Diluted cord
blood (20 mls) was gently overlaid on 20 mls of Ficoll-Paque Plus
(Amersham Biosciences Cat #17-1440-03). The cells were spun at
900.times. gravity for 60 min. The buffy coat was removed with a
glass pipette and was washed twice with PBS. The cells were
resuspended in FCB media (Iscove's (Gibco) supplemented with 10%
human plasma, 100 units per ml Penn/Strep, 30 ml of media
containing SCF, IL3 and IL6 and 30 mls of medium containing TPO,
FLT3-L, and GM-CSF described above in Example 1).
[0345] Two FCB expansion cultures were initiated. The first culture
contained FCB medium alone while the second contained FCB media
supplemented with 5 .mu.g/ml recombinant Tat-MYC, and 10 .mu.g/ml
recombinant Tat-Bcl-2. The medium on both cultures was replaced
every 3 days over a 14 day expansion. The surface phenotype of the
in vitro expanded human HSCs was assessed by FACS analysis using
antibodies against the human antigens CD45, CD34 and CD38.
[0346] Fetal cord blood cells (FCBs) expanded in FCB media or FCB
media supplemented with Tat-MYC and Tat-Bcl2 were injected into
NOD/SCID/.gamma.c-/- mice (NSG) mice (Jackson Laboratory) that
received 180 Rads of radiation just prior to injection. Expanded
FCBs were washed 3 times in PBS and injected via the tail vein in
200 .mu.l PBS. Eight weeks post-transplant, the bone marrow cells
were collected from the tibia and femur bones of the transplant NSG
mice. The red blood cells were lysed by incubation in 5 ml sterile
TAC buffer (135 mM NH.sub.4CL, 17 mM Tris Ph 7.65) followed by 2
washes D10 media. The BM cells were analyzed by flow cytometry
using antibodies to the human antigens CD45, CD34, CD38, CD3, CD19,
CD11b and CD33.
[0347] The spleen and thymus were collected from a euthanized NSG
mice and a single cell suspension was generated by mechanical
dissociation. The cells were treated with TAC buffer (135 mM
NH.sub.4CL, 17 mM Tris Ph 7.65) to lyse the red blood cells.
[0348] We functionally tested the human HSCs harvested from the BM
of NSG mice by plating them on MethoCult Optimum (StemCell
Technologies), and examined their ability to give rise to BFU-E,
CFU-M, CFU-G and CFU-GM colonies. On the day of the BM harvest,
10,000 BM cells in 300 ul D10 were added to 4 mls of MethoCult. The
4 ml containing the BM cells were divided equally between 2, 30 mM
dishes, each containing 5000 cells. The dishes were incubated at 37
in 5% CO.sub.2 for 14 days. The colonies were counted and
identified based on cell morphology using an inverted
microscope.
[0349] Results
[0350] As shown in FIG. 17, xenochimaeric NSG mice generated by
transplanting HSC expanded in FCB media supplemented with Tat-MYC
and Tat-Bcl2 (ptlt-HSCs) showed an increase in BM engraftment.
Eight weeks post-transplant the NSG mice injected with
1.times.10.sup.6 CD34+/CD38lo cells expanded in FCB media alone had
0.03% of their bone marrow compartment derived from the
transplanted cells (FIG. 17A, first panel). NSG mice injected with
1.times.10.sup.6 CD34+/CD38lo cells expanded in FCB media
supplemented with Tat-MYC and Tat-Bcl2 had 18.2% of their bone
marrow compartment derived from the transplanted cells (FIG. 17A,
second panel). An NSG mouse transplanted with 5.times.10.sup.6
fresh cord blood cells was used as a control for engraftment (FIG.
17A, third panel), and showed 2.7% engraftment with transplanted
cells.
[0351] Human CD45+ cells from the BM, spleen and thymus of
xenochimaeric NSG mice generated by transplanting HSC expanded in
FCB media supplemented with Tat-MYC and Tat-Bcl2 were analyzed.
FACS analysis shows that 6.8% of the human CD45+ population in the
BM also stained positive for the hematopoietic stem cell marker
CD34 (FIG. 17B, first panel). Human CD45+ cells from the spleen and
thymus of these mice were assessed for the B cell marker CD19 and
the T cell marker CD3. The majority of the human CD45+ cells from
the spleen stained positive for the B cell marker CD19 (FIG. 17B,
second panel, 69.2%), compared to the T cell marker CD3 (FIG. 17B,
second panel, 7.7%). The majority of CD45+ population in the thymus
stained positive for the CD3 T cell marker (FIG. 17B, third panel,
91.3%), compared to the B cell marker CD19 (FIG. 17B, third panel,
1.2%).
[0352] Human CD45+CD19+ cells from the spleens of xenochimaeric NSG
mice transplanted with ptlt-HSCs were labeled with CFSE, and were
activated with monoclonal antibodies to human CD40 and IgM. The
cells were analyzed at 72 hours by flow cytometry for dilution of
CFSE. FIG. 17C shows the proliferation profile of the human B-cells
that developed in vivo in xenochimaeric NSG mice. These results
demonstration that the B cells derived from the transplanted HSCs
that received pretreatment with Tat-MYC and Tat-Bcl2 can be
activated through their B cell receptor.
[0353] Human CD45+, CD34+CD38lo HSCs from the bone marrow of
xenochimaeric NSG mice transplanted with ptlt-HSCs were used to
seed in MethoCult Optimum to assess the presence of myeloerythroid
stem cells. These cells from NSG mice transplanted with ptlt-HSCs
gave rise to more colonies in MethoCult plates (FIG. 17D, FCB
TMTB), as compared to cells from control NSG mice transplanted with
cells expanded in media alone (FIG. 17D, FCB). Although colony
formation was observed in conditions selective for erythroid
(BFU-E), myeloid (CFU-M), granulocyte (CFU-G), and granulocyte
macrophage (CFU-GM) lineages, more myeloid and granulocyte growth
was observed. In addition, some of the colonies could still be
observed following serial replating (FIG. 17E). The number of
colonies in both instances was significantly higher for NSG mice
reconstituted with human cord blood cells cultured for 14 days with
Tat-MYC and Tat-Bcl-2 than for cells obtained from NSG mice
reconstituted with fresh, un-manipulated human cord blood
cells.
[0354] These results show that the CD45+, CD34+CD38lo HSCs from the
bone marrow of engrafted mice are hematopoietic stems cell that are
able to give rise to all 4 colony types in MethoCult media.
Further, the increased colony number observed on plates seeded with
bone marrow from NSG mice engrafted with FCBs cultures with Tat-MYC
and Tat-Bcl2 is reflective of a greater number of hematopoietic
stems cells residing in the bone marrow niche.
[0355] In addition, a cohort of xenochimaeric mice, engrafted with
10.sup.6 cord blood cells previously expanded in vitro in a
cocktail of cytokines supplemented with Tat-MYC and Tat-Bcl-2
(black squares), were assessed for myeloid and lymphoid cell
differentiation. The CD45 positive population of bone marrow cells
(FIG. 17F) and spleen cells (FIG. 17G) were analyzed for CD11b,
CD33, CD3, and CD19 expression. Both myeloid and lymphoid cell
differentiation was observed in the bone marrow and spleen of these
xenochimaeric mice.
Example 7: Accelerated Hematopoietic Reconstitution in Mice
Transplanted with Human Freshly Isolated Whole Cord Blood and
Injected with TAT-MYC
[0356] The following example describes the results of treating mice
with a TAT-MYC fusion protein after transplantation with fresh
human cord blood cells.
[0357] Materials and Methods
[0358] Human cord blood cells were obtained, and prepared as
described in Example 6. Briefly, the cord blood was separated using
Ficoll-hypaque and centrifugation to obtain the buffy coat
fraction. Buffy coat cells were removed, washed twice, then
resuspended in PBS, and kept cold until transplanted into mice
later the same day.
[0359] Prior to injecting NOD/SCID/.gamma.c.sup.-/- (NOG) mice with
FCB cells, the mice were irradiated with 180 Rads (whole body
irradiation). Each recipient mouse was then given a transplant
consisting of 5.times.10.sup.5, 1.times.10.sup.6, or
5.times.10.sup.6 human FCB cells via tail vein injection. 24 hours
after the bone marrow cell transplant, mice received an
intramuscular injection of 10 .mu.g of TAT-MYC or 10 .mu.g TAT-CRE
emulsified in 300 .mu.l of corn oil.
[0360] Engraftment was monitored with flow cytometry to assess for
the presence of human CD45 positive cells in the peripheral blood,
bone marrow, and spleen of the xenochimeric mice eight weeks after
transplantation.
[0361] Results
[0362] As shown in FIGS. 18, 19 and 20, accelerated development of
human cells was seen in the peripheral blood (FIG. 18), bone marrow
(FIG. 19), and spleen (FIG. 20) of xenochimeric
NOD/SCID/.gamma.c.sup.-/- mice treated with TAT-MYC following
xenotransplantation of human fresh fetal cord blood cells. Cohorts
of NOD/SCID/.gamma.c.sup.-/- mice were given a sub-lethal dose of
radiation followed by 5.times.10.sup.5 cord blood cells (FIGS. 18A
and 18D; FIGS. 19A and 19D; FIGS. 20A and 20D), 1.times.10.sup.6
(FIGS. 18B and 18E; FIGS. 19B and 19E; FIGS. 20B and 20E), or
5.times.10.sup.6 (FIGS. 18C and 18F; FIGS. 19C and 19E; FIGS. 20C
and 20E). 24 hours after the transplant, half of the mice in each
cohort were injected with TAT-MYC (FIGS. 18D, 18E, and 18F; FIGS.
19D, 19E, and 19F; FIGS. 20D, 20E, and 20F) and the other half were
injected with a control protein, TAT-CRE (FIGS. 18A, 18B, and 18C;
FIGS. 19A, 19B, and 19C; FIGS. 20A, 20B, and 20C)
[0363] FIG. 18 shows that control, sub-lethally irradiated
NOD/SCID/.gamma.c.sup.-/- mice, treated with TAT-CRE following
xenotransplantation with human whole cord blood cells, had in their
peripheral blood at eight weeks, 0% of CD45.sup.+ cells after
transplantation of 5.times.10.sup.5 cells (FIG. 18A), 0.2% of
CD45.sup.+ cells (FIG. 18B) after transplantation of
1.times.10.sup.6 cells, and 0.7% of CD45.sup.+ cells (FIG. 18C)
after transplantation of 5.times.10.sup.6 cells. Sub-lethally
irradiated NOD/SCID/.gamma.c.sup.-/- mice treated with TAT-MYC
following xenotransplantation with human whole cord blood cells,
had in their peripheral blood at eight weeks, 0.09% of CD45.sup.+
cells after transplantation of 5.times.10.sup.5 cells (FIG. 18D),
0.1% of CD45.sup.+ cells (FIG. 18E) after transplantation of
1.times.10.sup.6 cells, and 9.3% of CD45.sup.+ cells (FIG. 18F)
after transplantation of 5.times.10.sup.6 cells. Accordingly, FIG.
18F shows the detection of human T-cells (hCD45 positive cells) in
the peripheral blood of chimeric mice 8 weeks post-transplant. As
the average time to T-cell recovery is 12-20 weeks, this represents
an acceleration of about 33-60%.
[0364] FIG. 19 shows that control, sub-lethally irradiated
NOD/SCID/.gamma.c.sup.-/- mice, treated with TAT-CRE following
xenotransplantation with human whole cord blood cells, had in their
bone marrow at eight weeks, 0.02% of CD45.sup.+ cells after
transplantation of 5.times.10.sup.5 cells (FIG. 19A), 7.7% of
CD45.sup.+ cells (FIG. 19B) after transplantation of
1.times.10.sup.6 cells, and 11.3% of CD45.sup.+ cells (FIG. 19C)
after transplantation of 5.times.10.sup.6 cells. Sub-lethally
irradiated NOD/SCID/.gamma.c.sup.-/- mice treated with TAT-MYC
following xenotransplantation with human whole cord blood cells,
had in their bone marrow at eight weeks, 0.04% of CD45.sup.+ cells
after a transplantation of 5.times.10.sup.5 cells (FIG. 19D), 8.6%
of CD45.sup.+ cells (FIG. 19E) after a transplantation of
1.times.10.sup.6 cells, and 20.7% of CD45.sup.+ cells (FIG. 19F)
after a transplantation of 5.times.10.sup.6 cells.
[0365] FIG. 20 shows that control, sub-lethally irradiated
NOD/SCID/.gamma.c.sup.-/- mice, treated with TAT-CRE following
xenotransplantation with human whole cord blood cells, had in their
spleen at eight weeks, 0.9% of CD45.sup.+ cells after
transplantation of 5.times.10.sup.5 cells (FIG. 20A), 13.9% of
CD45.sup.+ cells (FIG. 20B) after transplantation of
1.times.10.sup.6 cells, and 27.6% of CD45.sup.+ cells (FIG. 20C)
after transplantation of 5.times.10.sup.6 cells. Sub-lethally
irradiated NOD/SCID/.gamma.c.sup.-/- mice treated with TAT-MYC
following xenotransplantation with human whole cord blood cells,
had in their spleen at eight weeks, 1.2% of CD45.sup.+ cells after
transplantation of 5.times.10.sup.5 cells (FIG. 20D), 4.9% of
CD45.sup.+ cells (FIG. 20E) after transplantation of
1.times.10.sup.6 cells, and 258% of CD45.sup.+ cells (FIG. 20F)
after transplantation of 5.times.10.sup.6 cells.
Example 8: Accelerated Hematopoietic Reconstitution in Mice
Transplanted with Human Freshly Isolated Cord Blood Cells
Pretreated with TAT-MYC and TAT-Bcl-2
[0366] The following example describes the results of pre-treating
fresh human cord blood cells with TAT-MYC and TAT-Bcl-2 prior to
transplantation into mice.
[0367] Materials and Methods
[0368] Human cord blood cells were obtained and prepared as
described in Example 6. Briefly, the cord blood was separated using
Ficoll-hypaque and centrifugation to obtain the buffy coat
fraction. Buffy coat cells were removed, washed twice, and then
resuspended in PBS.
[0369] Prior to injection into mice, the isolated cord blood cells
were exposed to 5 .mu.g/ml TAT-MYC and 5 .mu.g/ml TAT-Bcl-2 for one
hour. After exposure to the fusion proteins, the cells were washed
twice with PBS, then resuspended in PBS at 5.times.10.sup.6 cells
per 200 ul, and kept cold until injected into mice.
[0370] Prior to injecting NOD/SCID/.gamma.c.sup.-/- (NOG) mice with
the FCB cells, the mice were irradiated with 180 Rads (whole body
irradiation). Each recipient mouse was then given a transplant
consisting of 5.times.10.sup.6 human FCB cells in 200 ul PBS via
tail vein injection.
[0371] Engraftment was monitored with flow cytometry to assess the
presence of CD45 positive cells in the peripheral blood of the
xenochimeric mice. The first bleed was done 8 weeks after the FCB
transplant.
[0372] After eight months, the mice were euthanized and the bone
marrow cells were collected from the tibia and femur bones of the
xenochimaeric NSG mice. The spleen and thymus were also harvested,
and were made into single cell suspensions by pressing the cells
through a sterile wire mesh screen. The red blood cells from the
BM, spleen and thymus were lysed in 5 ml sterile TAC buffer (135 mM
NH.sub.4CL, 17 mM Tris Ph 7.65) followed by 2 washes in D10 media.
The BM, spleen cells and thymus cells were prepared for FACs
analysis to assess the presence of human CD45 positive cells.
[0373] Results
[0374] FIGS. 21 and 22 show that pre-treatment of human fresh whole
cord blood cells with TAT-MYC for one hour prior to transplantation
in NOD/SCID/.gamma.c.sup.-/- (NOG) mice resulted in accelerated
development of human CD45 cells in the peripheral blood of the
mice, as well as increased longterm persistence of human CD45 cells
in the bone marrow, spleen and thymus of the mice. Cohorts of
NOD/SCID/.gamma.c.sup.-/- mice were given a sub-lethal dose of
radiation followed by 5.times.10.sup.6 cord blood cells. For half
of the mice in each cohort, the cells were pre-treated with 5
.mu.g/ml TAT-MYC and 5 .mu.g/ml TAT-Bcl-2 in FCB media (FIG. 21B;
FIGS. 22B, 22D, and 22F); for the other half of the mice in the
cohort, the cells were pre-treated in FCB media alone (FIG. 21A;
FIGS. 22A, 22C, and 22E).
[0375] FIG. 21 shows that control, sub-lethally irradiated
NOD/SCID/.gamma.c.sup.-/- mice, xenotransplanted with human whole
cord blood cells pre-treated with FCB media alone, had in their
peripheral blood at eight weeks, 0.89% CD45.sup.+ cells (FIG. 21A).
In contrast, sub-lethally irradiated NOD/SCID/.gamma.c.sup.-/-
mice, xenotransplanted with human whole cord blood cells
pre-treated with 5 .mu.g/ml TAT-MYC and 5 .mu.g/ml TAT-Bcl-2 in FCB
media, had in their peripheral blood at eight weeks, 15.7%
CD45.sup.+ cells (FIG. 21B).
[0376] FIG. 22 shows that control, sub-lethally irradiated
NOD/SCID/.gamma.c.sup.-/- mice, xenotransplanted with human whole
cord blood cells pre-treated with FCB media alone, had at eight
months in their bone marrow, 0.04% CD45.sup.+ cells (FIG. 22A), in
their spleen 0.03% CD45.sup.+ cells (FIG. 22C), and in their
thymus, 0.5% CD45.sup.+ cells (FIG. 22E). In contrast, sub-lethally
irradiated NOD/SCID/.gamma.c.sup.-/- mice, xenotransplanted with
human whole cord blood cells pre-treated with 5 .mu.g/ml TAT-MYC
and 5 .mu.g/ml TAT-Bcl-2 in FCB media, had at eight months in their
bone marrow, 0.37% CD45.sup.+ cells (FIG. 22B), in their spleen
25.2% CD45.sup.+ cells (FIG. 22D), and in their thymus, 5.4%
CD45.sup.+ cells (FIG. 22F).
[0377] In other experiments, 5 .mu.g/ml TAT-MYC and 5 .mu.g/ml
TAT-Bcl-2 are used to pre-treat separate populations of human cord
blood cells prior to being transplanted into different cohorts of
NOG mice. The inventors expect that separate incubation of cells
with TAT-MYC and TAT-Bcl-2 will still afford better engraftment and
reconstitution results than transplantation of the same cells
without pre-treatment.
Example 9: Accelerated Engraftment with Human G-CSF Mobilized
Peripheral Blood HSCs Cultured with Tat-Myc and Tat-Bcl-2
[0378] G-CSF mobilized cells were received in a 1 ml volume of
elutriated blood from 5 patients who underwent G-CSF mobilization
for autologous HSC transplantation. All G-CSF samples were
de-identified and no further identifying information is associated
with the cells used for these studies. The cells were added drop
wise to 10 ml of FCB media. The cells were washed twice in FCB
media and treated with 5 .mu.g/ml recombinant Tat-Myc and 5
.mu.g/ml recombinant Tat-Bcl-2 in a 10 ml volume. Cells were
cultured for 12 days
[0379] The cells were expanded in media supplemented with cytokines
plus Tat-Myc and Tat-Bcl2 for 12 days. On the 12.sup.th day half of
the cells received an additional treatment with 5 .mu.g/ml Tat-MYC
and 5 .mu.g/ml Tat-Bcl2. The other half of the cells were left in
FCB media alone. The cells were incubated for 60 minutes in a 37
incubator. The cells were washed three times in PBS, and were
resuspended at 5.times.10.sup.6 cells per 200 ul. Engraftment was
monitored with flow cytometry to assess the presence of CD45
positive cells in the peripheral blood of the xenochimeric mice.
The first bleed was done 8 weeks after the HSC transplant.
[0380] FIG. 23A shows a FACS analysis of the CD45.sup.+ staining of
the peripheral blood from control NSG (FIG. 23A), NSG mice
transplanted 8 weeks earlier with either 1.times.10.sup.6 expanded
G-CSF mobilized HSCs (FIG. 23B) or 1.times.10.sup.6 expanded G-CSF
mobilized HSC treated with Tat-Myc/Tat-Bcl-2 (FIG. 23C). As shown
for the peripheral blood, the inventors expect that the BM, spleen
and thymus from mice engrafted with G-CSF mobilized cells
pretreated with TAT-MYC and TAT-Bcl-2 will afford better
engraftment and reconstitution results than transplantation of the
same cells without pre-treatment.
Example 10: Accelerated Hematopoietic Reconstitution in Mice
Treatment with TAT-MYC Following Transplantation with Human G-CSF
Mobilized Cells Pretreated with TAT-NYC and TAT-Bcl-2
[0381] The following example describes the results of pre-treating
human G-CSF mobilized cells with TAT-MYC and TAT-Bcl-2 prior to
transplantation into mice, and then followed by treatment of the
mice with TAT-MYC 24 hours later.
[0382] Materials and Methods
[0383] G-CSF mobilized cells were received in a 1 ml volume of
elutriated blood from 5 patients who underwent G-CSF mobilization
for autologous HSC transplantation. All G-CSF samples were
de-identified and no further identifying information was associated
with the cells used for these studies. The cells were added drop
wise to 10 ml of FCB media. The cells were washed twice in FCB
media and treated with 5 .mu.g/ml recombinant Tat-Myc and 5
.mu.g/ml recombinant Tat-Bcl-2 in a 10 ml volume. The cells were
cultured for 12 days.
[0384] On Day 12, the expanded G-CSF mobilized cells received a
second treatment of 5 .mu.g/ml TAT-MYC and 5 .mu.g/ml TAT-Bcl2 for
hour prior to injecting into NSG mice. The cells were washed 3
times with PBS and then injected at 5.times.10.sup.6 cell per mouse
in 200 .mu.l PBS via the tail vein. Prior to injecting
NOD/SCID/.gamma.c.sup.-/- (NOG) mice with the HSC cells, the mice
were irradiated with 180 Rads (whole body irradiation). 24 hours
post injection with the expanded HSCs, the mice received 10 .mu.g
of Tat-MYC or 10 .mu.g Tat-Cre in corn oil intramuscularly, or no
injection. Eight weeks later the mice were bled.
[0385] Engraftment was monitored with flow cytometry to assess the
presence of CD45 positive cells in the peripheral blood of the
xenochimeric mice. The first bleed was done 8 weeks after the HSC
transplant.
[0386] After eight weeks, the mice are euthanized and the bone
marrow cells are collected from the tibia and femur bones of the
xenochimaeric NSG mice. The spleen and thymus are also harvested,
and are made into single cell suspensions by pressing the cells
through a sterile wire mesh screen. The red blood cells from the
BM, spleen and thymus are lysed in 5 ml sterile TAC buffer (135 mM
NH.sub.4CL, 17 mM Tris Ph 7.65) followed by 2 washes in D10 media.
The BM, spleen cells and thymus cells are prepared for FACs
analysis to assess the presence of human CD45 positive cells.
[0387] Results
[0388] The data indicate that injection of TAT-MYC 24 hours after
transplantation of HSCs pre-treated with TAT-MYC and TAT-Bcl-2 for
one hour prior to transplantation in NOD/SCID/.gamma.c.sup.-/-
(NOG) mice did not appear to accelerate development of human CD45
cells in the peripheral blood of the mice, as compared with control
mice injected with TAT-CRE or with no Tat-fusion protein injection,
but also transplanted with HSCs pre-treated with TAT-MYC and
TAT-Bcl-2.
[0389] At eight weeks, the data show no difference between mice
treated with TAT-MYC and TAT-CRE in the levels of CD45+ cells in
the peripheral blood. Further investigation is needed to determine
if an effect exists at earlier or later time points, or in the BM,
spleen, and thymus. At this point, the data indicate that
pretreating the cells with TAT-MYC may work just as well as
injecting the mice with TAT-MYC after transplant. To date, we have
not observed an increased effect by pretreating the cells and also
injecting the mice with Tat-MYC after the transplant of treated
cells.
Example 11: Generation of Biologically Active Tat-Myc and Tat-Bcl-2
Fusion Proteins
[0390] Fusion proteins having the HIV-1 Tat protein transduction
domain (PTD) and either the ORF for human Myc, or a truncated form
of human Bcl-2, that has been deleted for the unstructured loop
domain (Anderson, M., et al. (1999). Prot Expr. Purif 15, 162-70),
were generated. The recombinant proteins also encoded a V5 peptide
tag and a 6-His tag (seq id no: 6), to facilitate detection and
purification (FIG. 24 and FIG. 25).
[0391] pTAT-Myc-V5-6.times.His (Amp.sup.R) and
pTAT-Bcl2.DELTA.-V5-6.times.His(Amp.sup.R): plasmid were generated
by PCR amplification of a cDNA encoding human cMyc or human Bcl2
using a forward primer encoding an in frame TAT protein
transduction domain of HIV (RKKRRQRRR) (SEQ ID NO: 5). The PCR
products were cloned into pET101/D-Topo (Invitrogen) vector. The
unstructured loop (A.A. #27-80) was removed from the BCL-2 coding
sequence using a Quick Change site directed mutagenesis kit
(Stratagene #200521-5).
[0392] The proteins were synthesized in E. coli and purified to
homogeneity. SDS-PAGE electrophoresis and Coomassie Staining
revealed the level of purity of the final product used for our
studies (FIG. 1B). pTAT-Myc-V5-6.times.His was transformed into
BL21-STAR(DE3) cells (Invitrogen) and protein was induced with 0.5
mM IPTG at 37.degree. C. for 3 hrs. The cells were lysed in lysis
buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris pH to 7.0, 10 mM
imidazole, pH 7.2). The lysate was diluted to 6M urea and brought
to 450 mM NaCl, 50 mM NaH.sub.2PO.sub.4, 5 mM Tris pH 7.0. The
lysate was treated with Benzonase (500 units) at room temp for 1
hour, clarified by centrifugation at 12,000 RPM for 60 min and
filtered through a 0.22 .mu.M filter. Myc-V5-6.times.His was
purified on a nickel affinity column (GE) using a GE AKTA purifier
10 FPLC. Myc-V5-6.times.His was refolded by dialyzing into dialysis
buffer (450 mM NaCl, 50 mM NaH.sub.2PO.sub.4, 5 mM Tris pH 7.0, 5%
glycerol, 1 mM DTT). Endotoxin was reduced by passing the purified
protein over an Acticlean Etox column (Sterogen).
[0393] Bcl2A-V5-6.times.His protein was induced as described above.
The cells were lysed in 50 mL of lysis buffer (200 mM NaCl, 200 mM
KCL, 50 mM NaH.sub.2PO.sub.4, 5 mM Tris pH 7.0, 5% glycerol, 1 mM
DTT) supplemented with 500 units Benzonase, 1 mM PMSF, 2 .mu.g/ml
Leupeptin, 0.015 units/ml Aprotinin, 5 uM Hen Egg Lysozyme (HEL)
per 1 L of induced protein, and immediately placed on ice for 1
hour. The cells were sonicated on ice (Duty cycle=50%, Output=5)
for 2 sets of 2 minutes. The lysate was cleared by centrifugation
at 12,000 RPM for 60 min and was filtered through a 0.22 .mu.M
filter. Bcl2A-V5-6.times.His was purified on a nickel affinity
column (GE) and endotoxin was removed as described above.
Sequence CWU 1
1
81477PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Met Arg Lys Lys Arg Arg Gln Arg Arg Arg Met
Pro Leu Asn Val Ser1 5 10 15Phe Thr Asn Arg Asn Tyr Asp Leu Asp Tyr
Asp Ser Val Gln Pro Tyr 20 25 30Phe Tyr Cys Asp Glu Glu Glu Asn Phe
Tyr Gln Gln Gln Gln Gln Ser 35 40 45Glu Leu Gln Pro Pro Ala Pro Ser
Glu Asp Ile Trp Lys Lys Phe Glu 50 55 60Leu Leu Pro Thr Pro Pro Leu
Ser Pro Ser Arg Arg Ser Gly Leu Cys65 70 75 80Ser Pro Ser Tyr Val
Ala Val Thr Pro Phe Ser Leu Arg Gly Asp Asn 85 90 95Asp Gly Gly Gly
Gly Ser Phe Ser Thr Ala Asp Gln Leu Glu Met Val 100 105 110Thr Glu
Leu Leu Gly Gly Asp Met Val Asn Gln Ser Phe Ile Cys Asp 115 120
125Pro Asp Asp Glu Thr Phe Ile Lys Asn Ile Ile Ile Gln Asp Cys Met
130 135 140Trp Ser Gly Phe Ser Ala Ala Ala Lys Leu Val Ser Glu Lys
Leu Ala145 150 155 160Ser Tyr Gln Ala Ala Arg Lys Asp Ser Gly Ser
Pro Asn Pro Ala Arg 165 170 175Gly His Ser Val Cys Ser Thr Ser Ser
Leu Tyr Leu Gln Asp Leu Ser 180 185 190Ala Ala Ala Ser Glu Cys Ile
Asp Pro Ser Val Val Phe Pro Tyr Pro 195 200 205Leu Asn Asp Ser Ser
Ser Pro Lys Ser Cys Ala Ser Gln Asp Ser Ser 210 215 220Ala Phe Ser
Pro Ser Ser Asp Ser Leu Leu Ser Ser Thr Glu Ser Ser225 230 235
240Pro Gln Gly Ser Pro Glu Pro Leu Val Leu His Glu Glu Thr Pro Pro
245 250 255Thr Thr Ser Ser Asp Ser Glu Glu Glu Gln Glu Asp Glu Glu
Glu Ile 260 265 270Asp Val Val Ser Val Glu Lys Arg Gln Ala Pro Gly
Lys Arg Ser Glu 275 280 285Ser Gly Ser Pro Ser Ala Gly Gly His Ser
Lys Pro Pro His Ser Pro 290 295 300Leu Val Leu Lys Arg Cys His Val
Ser Thr His Gln His Asn Tyr Ala305 310 315 320Ala Pro Pro Ser Thr
Arg Lys Asp Tyr Pro Ala Ala Lys Arg Val Lys 325 330 335Leu Asp Ser
Val Arg Val Leu Arg Gln Ile Ser Asn Asn Arg Lys Cys 340 345 350Thr
Ser Pro Arg Ser Ser Asp Thr Glu Glu Asn Val Lys Arg Arg Thr 355 360
365His Asn Val Leu Glu Arg Gln Arg Arg Asn Glu Leu Lys Arg Ser Phe
370 375 380Phe Ala Leu Arg Asp Gln Ile Pro Glu Leu Glu Asn Asn Glu
Lys Ala385 390 395 400Pro Lys Val Val Ile Leu Lys Lys Ala Thr Ala
Tyr Ile Leu Ser Val 405 410 415Gln Ala Glu Glu Gln Lys Leu Ile Ser
Glu Glu Asp Leu Leu Arg Lys 420 425 430Arg Arg Glu Gln Leu Lys His
Lys Leu Glu Gln Leu Arg Lys Gly Glu 435 440 445Leu Asn Ser Lys Leu
Glu Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly 450 455 460Leu Asp Ser
Thr Arg Thr Gly His His His His His His465 470
47521299DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 2atgaggaaga agcggagaca gcgacgaaga
atgcccctca acgttagctt caccaacagg 60aactatgacc tcgactacga ctcggtgcag
ccgtatttct actgcgacga ggaggagaac 120ttctaccagc agcagcagca
gagcgagctg cagcccccgg cgcccagcga ggatatctgg 180aagaaattcg
agctgctgcc caccccgccc ctgtccccta gccgccgctc cgggctctgc
240tcgccctcct acgttgcggt cacacccttc tcccttcggg gagacaacga
cggcggtggc 300gggagcttct ccacggccga ccagctggag atggtgaccg
agctgctggg aggagacatg 360gtgaaccaga gtttcatctg cgacccggac
gacgagacct tcatcaaaaa catcatcatc 420caggactgta tgtggagcgg
cttctcggcc gccgccaagc tcgtctcaga gaagctggcc 480tcctaccagg
ctgcgcgcaa agacagcggc agcccgaacc ccgcccgcgg ccacagcgtc
540tgctccacct ccagcttgta cctgcaggat ctgagcgccg ccgcctcaga
gtgcatcgac 600ccctcggtgg tcttccccta ccctctcaac gacagcagct
cgcccaagtc ctgcgcctcg 660caagactcca gcgccttctc tccgtcctcg
gattctctgc tctcctcgac ggagtcctcc 720ccgcagggca gccccgagcc
cctggtgctc catgaggaga caccgcccac caccagcagc 780gactctgagg
aggaacaaga agatgaggaa gaaatcgatg ttgtttctgt ggaaaagagg
840caggctcctg gcaaaaggtc agagtctgga tcaccttctg ctggaggcca
cagcaaacct 900cctcacagcc cactggtcct caagaggtgc cacgtctcca
cacatcagca caactacgca 960gcgcctccct ccactcggaa ggactatcct
gctgccaaga gggtcaagtt ggacagtgtc 1020agagtcctga gacagatcag
caacaaccga aaatgcacca gccccaggtc ctcggacacc 1080gaggagaatg
tcaagaggcg aacacacaac gtcttggagc gccagaggag gaacgagcta
1140aaacggagct tttttgccct gcgtgaccag atcccggagt tggaaaacaa
tgaaaaggcc 1200cccaaggtag ttatccttaa aaaagccaca gcatacatcc
tgtccgtcca agcagaggag 1260caaaagctca tttctgaaga ggacttgttg
cggaaacga 12993235PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 3Met Arg Lys Lys Arg Arg Gln Arg Arg
Arg Met Ala His Ala Gly Arg1 5 10 15Ser Gly Tyr Asp Asn Arg Glu Ile
Val Met Lys Tyr Ile His Tyr Lys 20 25 30Leu Ser Gln Arg Ala Thr Ser
Gly Ile Ser Ile Glu Ala Ala Gly Pro 35 40 45Ala Leu Ser Pro Val Pro
Pro Val Val His Leu Thr Leu Arg Gln Ala 50 55 60Gly Asp Asp Phe Ser
Arg Arg Tyr Arg Arg Asp Phe Ala Glu Met Ser65 70 75 80Ser Gln Leu
His Leu Thr Pro Phe Thr Ala Arg Gly Cys Phe Ala Thr 85 90 95Val Val
Glu Glu Leu Phe Arg Asp Gly Val Asn Trp Gly Arg Ile Val 100 105
110Ala Phe Phe Glu Phe Gly Gly Val Met Cys Val Glu Ser Val Asn Arg
115 120 125Glu Met Ser Pro Leu Val Asp Asn Ile Ala Leu Trp Met Thr
Glu Tyr 130 135 140Leu Asn Arg His Leu His Thr Trp Ile Gln Asp Asn
Gly Gly Trp Asp145 150 155 160Ala Phe Val Glu Leu Tyr Gly Pro Ser
Met Arg Pro Leu Phe Asp Phe 165 170 175Ser Trp Leu Ser Leu Lys Thr
Leu Leu Ser Leu Ala Leu Val Gly Ala 180 185 190Cys Ile Thr Leu Gly
Ala Tyr Leu Ser His Lys Lys Gly Glu Leu Asn 195 200 205Ser Lys Leu
Glu Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp 210 215 220Ser
Thr Arg Thr Gly His His His His His His225 230 2354708DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
4atgaggaaga agcggagaca gcgacgaaga atggcgcacg ctgggagaag tggttacgat
60aaccgggaga tagtgatgaa gtacatccat tataagctgt cgcagagggc tacgagtggg
120atctcgatcg aggccgcggg gcctgcgctc agcccggtgc cacctgtggt
ccacctgacc 180ctccgccagg ccggcgacga cttctcccgc cgctaccgcc
gcgacttcgc cgagatgtcc 240agccagctgc acctgacgcc cttcaccgcg
cggggatgct ttgccacggt ggtggaggag 300ctcttcaggg acggggtgaa
ctgggggagg attgtggcct tctttgagtt cggtggggtc 360atgtgtgtgg
agagcgtcaa ccgggagatg tcgcccctgg tggacaacat cgccctgtgg
420atgactgagt acctgaaccg gcacctgcac acctggatcc aggataacgg
aggctgggat 480gcctttgtgg aactgtacgg ccccagcatg cggcctctgt
ttgatttctc ctggctgtct 540ctgaagactc tgctcagttt ggccctggtg
ggagcttgca tcaccctggg tgcctatctg 600agccacaaga agggcgagct
caattcgaag cttgaaggta agcctatccc taaccctctc 660ctcggtctcg
attctacgcg taccggtcat catcaccatc accattga 70859PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Arg
Lys Lys Arg Arg Gln Arg Arg Arg1 566PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 6xHis tag 6His
His His His His His1 5714PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 7Gly Lys Pro Ile Pro Asn Pro
Leu Leu Gly Leu Asp Ser Thr1 5 1089PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 8Ile
Pro Asn Pro Leu Leu Gly Leu Asp1 5
* * * * *