Exhibit
10.1


AGREEMENT
BY AND BETWEEN REGEN BIOPHARMA, INC. AND SANTOSH KESARI 


Agreement
by and between Santosh Kesari (“Consultant”) , a natural person whose address is at 3525 Del Mar Heights Road #133,
San Diego CA 92130 and Regen Biopharma, Inc. (“Company”) , a Nevada corporation whose address is 4700 Spring Street,
St 304, La Mesa, California 91942. 


It
is agreed as follows:


1.
SCOPE OF SERVICES 



		(a)	Consultant
                                         shall complete all experiments, procedures, studies and tasks described within that document
                                         entitled “ Dr. Kesari Lab-Regen BioPharma Collaborative Project-dCellVax FDA Requested
                                         Studies” (“Proposal”) attached to this Agreement as EXHIBT A and incorporated
                                         herein.



		(b)	Consultant
                                         shall assist the Company in the preparation of an Investigational New Drug Application
                                         (“IND”) to be submitted to the United States Food and Drug Administration
                                         (“FDA”) with regard to the marketing of the Company’s proprietary product
                                         “DCellVax” as a treatment for gliomas such a assistance to be provided for
                                         a period of no less than twelve months from the execution date of this Agreement.



2. COMPENSATION 



		(a)	Consultant
                                         shall receive that number of common shares of the company, valued as of the closing price
                                         on the OTCBB as of the date of execution of this Agreement, which shall equal $66,000
                                         USD (“Signing Shares”). In the event that the date of execution of this Agreement
                                         is not a Trading Day then the Signing Shares shall be valued as of the closing price
                                         on the OTCBB as of the latest complete Trading Day immediately prior to the execution
                                         of this Agreement. “Trading Day” shall mean any day on which the common shares
                                         of the Company are tradable for any period on the OTBB.



		(b)	One
                                         half of the Signing Shares to be issued shall be registered under the Securities Act
                                         of 1933 on Form S-8.



		(c)	Signing
                                         Shares shall be issued to the Consultant on or prior to the expiration of a two week
                                         period following the execution of this Agreement.



		(d)	Upon
                                         completion of the studies described within Section 3 of the Proposal and successful demonstration
                                         of silencing of indolamine 2,3 deoxygenase in human dendritic cells Consultant shall
                                         be entitled to receive that number of common shares of the company, valued as of the
                                         closing price on the OTCBB as of the date that successful demonstration of silencing
                                         is presented to the Company by the Consultant (“Milestone Date”) , which
                                         shall equal $66,000 USD (“Milestone Shares”). In the event that the Milestone
                                         Date is not a Trading Day then the Milestone Shares shall be valued as of the closing
                                         price on the OTCBB as of the latest complete Trading Day immediately prior to the Milestone
                                         Date



		(e)	One
                                         half of the Milestone Shares to be issued shall be registered under the Securities Act
                                         of 1933 on Form S-8.



		(f)	Milestone
                                         Shares shall be issued to the Consultant on or prior to the expiration of a two week
                                         period following the Milestone Date.



		(g)	Upon
                                         the date of submission to the FDA of a response, prepared by the Consultant, providing
                                         evidence of vitro and/or in vivo confirmation of efficacy of the human siRNA sequences
                                         proposed for the clinical trial with regard to IND# 16200 for
                                         a proposed Phase I/II clinical trial assessing safety with signals of efficacy of the
                                         dCellVax gene silenced dendritic cell immunotherapy for treating breast cancer ( “Response
                                         Date”) Consultant shall be entitled to receive that number of common shares of
                                         the company, valued as of the closing price on the OTCBB as of the Response Date which
                                         shall equal $66,000 USD (“Response Date Shares”). In the event that the Response
                                         Date is not a Trading Day then the Response Date Shares shall be valued as of the closing
                                         price on the OTCBB as of the latest complete Trading Day immediately prior to the Response
                                         Date.



		(h)	One
                                         half of the Response Date Shares to be issued shall be registered under the Securities
                                         Act of 1933 on Form S-8.



		(i)	Response
                                         Date Shares shall be issued to the Consultant on or prior to the expiration of a two
                                         week period following the Response Date.



3. NON DISCLOSURE


		(a)	All
                                         information, whether in oral, written, graphic, electronic or other form, disclosed by
                                         the Company to the Consultant shall be deemed to be “Proprietary Information.”
                                         In particular, Proprietary Information includes, without limitation, any trade secrets,
                                         confidential information, ideas, inventions or research and development information;
                                         matters of a technical nature, including technology; notes, products, know-how, engineering
                                         or other data (including test data and data files); specifications, processes, techniques,
                                         formulae or work-in-process; manufacturing, planning or marketing procedures, clinical
                                         data and regulatory strategies or information; accounting, financial or pricing procedures
                                         or information, budgets or projections, or personnel or salary structure/compensation
                                         information; information regarding suppliers, clients, customers, employees, contractors,
                                         investors or investigators of the Company, information which has been designated in writing
                                         as confidential by the Company; programs, procedures (including operating procedures),
                                         processes, methods, guidelines, policies, proposals or contracts; computer software,
                                         data bases or programming; and any other information which, if divulged to a third party,
                                         could have an adverse impact on the Company, or on any third party to which it owes a
                                         confidentiality obligation. In addition, “Proprietary Information” includes
                                         any of the foregoing relating to the past, present or future operations, organization,
                                         projects, finances, business interests, methodology or affairs of any third party to
                                         which the Company owes a duty of confidentiality including, without limitation, the mere
                                         fact that the Company is or may be working with or for any client.




		(b)	The
                                         obligations of confidentiality shall not apply to any Proprietary Information that was
                                         known by the Consultant at the time of disclosure to it by such Company, or that is independently
                                         developed or discovered by the Consultant after disclosure by such Company, without the
                                         aid, application or use of any item of such Company’s Proprietary Information,
                                         as evidenced by written records; now, or subsequently becomes, through no act or failure
                                         to act on the part of the Consultant, generally known or available; is disclosed to the
                                         Consultant by a third party authorized to disclose it; or is required by law or by court
                                         or administrative order to be disclosed; provided, that the Consultant shall have first
                                         given prompt notice to such Company of such required disclosure.




		(c)	Consultant
                                         shall exercise due care to prevent the unauthorized use or disclosure of the Company’s
                                         Proprietary Information, and shall not, without the Company’s prior written consent,
                                         disclose or otherwise make available, directly or indirectly, any item of the Company’s
                                         Proprietary Information to any person or entity other than those employees, independent
                                         contractors or agents of the Consultant (collectively, “Representatives”),
                                         to the extent such Representatives reasonably need to know the same in order to evaluate
                                         such Proprietary Information, to participate in the business relationship between the
                                         parties, or to make decisions or render advice in connection therewith. Consultant shall
                                         advise its Representatives who have access to the Company’s Proprietary Information
                                         of the confidential and proprietary nature thereof, and agrees that such Representatives
                                         shall be bound by terms of confidentiality and restrictions on use with respect thereto
                                         that are at least as restrictive as the terms of this Agreement.



		(d)	Consultant
                                         shall exercise due care to prevent the unauthorized use or disclosure of the Company’s
                                         Proprietary Information, and shall not, without the Company’s prior written consent,
                                         disclose or otherwise make available, directly or indirectly, any item of the Company’s
                                         Proprietary Information to any person or entity other than those employees, independent
                                         contractors or agents of the Consultant (collectively, “Representatives”),
                                         to the extent such Representatives reasonably need to know the same in order to participate
                                         in any business relationship between the parties, or to make decisions or render advice
                                         in connection therewith. Consultant shall advise its Representatives who have access
                                         to the Company’s Proprietary Information of the confidential and proprietary nature
                                         thereof, and agrees that such Representatives shall be bound by terms of confidentiality
                                         and restrictions on use with respect thereto that are at least as restrictive as the
                                         terms of this Agreement.



		(e)	Consultant
                                         shall use the Company’s Proprietary Information solely for the purposes of performing
                                         his duties pursuant to this Agreement and shall not make any other use of the Company’s
                                         Proprietary Information without the Company’s specific written authorization.



		(f)	All
                                         Proprietary Information of the Company (including all copies thereof) shall be and at
                                         all times remain the property of such Company, and all non-oral Proprietary Information
                                         of the Company which is then in the Consultant’s possession or control shall be
                                         destroyed or returned to the Company promptly upon its request at any time, and in any
                                         event, no later than 60 days following any expiration or termination of this Agreement.



		(g)	Nothing
                                         in this Agreement shall be construed, by implication or otherwise, as a grant of any
                                         right or license to trademarks, inventions, copyrights or patents, as a grant of a license
                                         to either Consultant to use any of the Company’s Proprietary Information except
                                         as expressly set forth herein.



		(h)	The
                                         provisions of Section 3 of this Agreement shall survive until such time as all Confidential
                                         Information disclosed hereafter becomes publically known and made generally available
                                         through no action or inaction of Consultant.



 


4. REPRESENTATIONS AND WARRANTIES OF COMPANY


 


		(a)	Company
                                         is a corporation duly organized, validly existing and in good standing under the laws
                                         of the state its incorporation and has the requisite corporate power and authority to
                                         enter into and perform its obligations under this Agreement without the consent, approval
                                         or authorization of, or obligation to notify, any person, entity or governmental agency
                                         which consent has not been obtained.



		(b)	The
                                         execution, delivery and performance of this Agreement by Company does not and shall not
                                         constitute Company’s breach of any statute or regulation or ordinance of any governmental
                                         authority, and shall not conflict with or result in a breach of or default under any
                                         of the terms, conditions, or provisions of any order, writ, injunction, decree, contract,
                                         agreement, or instrument to which the Company is a party, or by which Company is or may
                                         be bound.





 
5. REPRESENTATIONS AND WARRANTIES OF CONSULTANT


 


		(a)	Consultant
                                         has the requisite power and authority to enter into and perform his obligations under
                                         this Agreement without the consent, approval or authorization of, or obligation to notify,
                                         any person, entity or governmental agency which consent has not been obtained.



		(b)	The
                                         execution, delivery and performance of this Agreement by Consultant does not and shall
                                         not constitute Consultant’s breach of any statute or regulation or ordinance of
                                         any governmental authority, and shall not conflict with or result in a breach of or default
                                         under any of the terms, conditions, or provisions of any order, writ, injunction, decree,
                                         contract, agreement, or instrument to which the Consultant is a party, or by which Company
                                         is or may be bound.





 


6. RESTRICTED SECURITIES
ACKNOWLEDGMENT



Consultant
acknowledges that any securities issued pursuant to this Agreement that shall not be registered pursuant to the Securities Act
of 1933 shall constitute “restricted securities” as that term is defined in Rule 144 promulgated under the Securities
Act of 1933, and shall contain the following restrictive legend:


“THESE
SECURITIES HAVE NOT BEEN REGISTERED UNDER THE SECURITIES ACT OF 1933, AS AMENDED (THE “ACT”), OR SECURITIES LAWS OF
ANY STATE AND MAY NOT BE OFFERED, SOLD, ASSIGNED, PLEDGED, TRANSFERRED OR OTHERWISE DISPOSED OF IN THE ABSENCE OF AN EFFECTIVE
REGISTRATION STATEMENT UNDER THE ACT AND APPLICABLE STATE SECURITIES LAWS OR PURSUANT TO AN AVAILABLE EXEMPTION FROM REGISTRATION
UNDER THE ACT OR SUCH LAWS AND, IF REQUESTED BY THE COMPANY, UPON DELIVERY OF AN OPINION OF COUNSEL REASONABLY SATISFACTORY TO
THE COMPANY THAT THE PROPOSED TRANSFER IS EXEMPT FROM THE ACT OR SUCH LAWS.” 



7.
SPECIFIC PERFORMANCE 


Any
breach of this Agreement may result in irreparable damage to Company for which Company will not have an adequate remedy at law.
Accordingly, in addition to any other remedies and damages available, Consultant acknowledges and agrees that Company may immediately
seek enforcement of this Agreement by means of specific performance or injunction, without any requirement to post a bond or other
security.


8.
EXECUTION 



This
Agreement may be executed in two or more counterparts, all of which when taken together shall be considered one and the same Agreement
and shall become effective when counterparts have been signed by each party and delivered to the other party, it being understood
that both parties need not sign the same counterpart. In the event that any signature is delivered by facsimile transmission,
such signature shall create a valid and binding obligation of the party executing (or on whose behalf such signature is executed)
with the same force and effect as if such facsimile signature page were an original thereof.


 9.
ENTIRE AGREEMENT 


This
Agreement constitutes a final written expression of all the terms of the Agreement between the parties regarding the subject matter
hereof, are a complete and exclusive statement of those terms, and supersedes all prior and contemporaneous Agreements, understandings,
and representations between the parties.


10. SEVERABILITY 



If
any provision of this Agreement is held to be invalid or unenforceable in any respect, the validity and enforceability of the
remaining terms and provisions of this Agreement shall not in any way be affected or impaired thereby and the parties will attempt
to agree upon a valid and enforceable provision that is a reasonable substitute therefore, and upon so agreeing, shall incorporate
such substitute provision in this Agreement


11.GOVERNING
LAW, VENUE, WAIVER OF JURY TRIAL


All
questions concerning the construction, validity, enforcement and interpretation of this Agreement shall be governed by and construed
and enforced in accordance with the internal laws of the State of California, without regard to the principles of conflicts of
law thereof. Each party hereby irrevocably submits to the exclusive jurisdiction of the state and federal courts sitting in California
for the adjudication of any dispute hereunder or in connection herewith or with any transaction contemplated hereby or discussed
herein and hereby irrevocably waives, and agrees not to assert in any suit, action or proceeding, any claim that it is not personally
subject to the jurisdiction of any such court, that such suit, action or proceeding is improper or inconvenient venue for such
proceeding. If either party shall commence an action or proceeding to enforce any provisions of this Agreement, then the prevailing
party in such action or proceeding shall be reimbursed by the other party for its attorneys’ fees and other costs and expenses
incurred with the investigation, preparation and prosecution of such action or proceeding.


IN
WITNESS WHEREOF, the parties hereto have caused this Agreement to be duly executed by their respective authorized signatories
as of the date first indicated above. 


	Company 	Consultant
	By:/s/ David R. Koos 	By:/s/Santosh Kesari 
	David R. Koos	Dr. Satosh Kesari
	Chairman & CEO 	
	Regen Biopharma, Inc. 	
	Date: June 8, 2015	Date: May 13, 2015 






    	 



    

 





EXHIBIT
A


 


Dr.
Kesari Lab-Regen BioPharma Collaborative Project


 


 


 


 


dCellVax
FDA Requested Studies


 


 


 


April
2015


 


 


 


 


Background


Regen
BioPharma has purchased a US patent and licensed Benitec Biopharma’s ddRNAi technology for the field of use of inhibiting
indolamine 2,3 deoxygenase (IDO) in dendritic cells (DC) for the purpose of immune stimulation in cancer patients.


Through
silencing of murine IDO in DC, Regen scientist Dr. Wei-Ping Min demonstrated inhibition of melanoma and breast cancer in the B16[1]
and 4T1 models[2], respectively.


Based
on these preclinical data, Regen BioPharma submitted an IND for treatment of breast cancer in patients that have failed conventional
therapy. The FDA responded to this IND requesting “in vitro and/or in vivo confirmation of efficacy of the human siRNA sequences
proposed for the clinical trial” before the clinical trial can be initiated.


The
experiments proposed below are designed to demonstrate in vitro efficacy of human IDO siRNA in the human DC in vitro model. Upon
completion of these experiments, the results will be submitte to the FDA for allowance to initiate clinical trials.


Objectives


		•	Development
                                         and optimization of sensitive molecular methods (reverse transcriptase polymerase chain
                                         reaction (RT-PCR)) for detecting the IDO gene, initially in human cancer cells[3],
                                         which then will be transferred to human dendritic cells



 


		•	Development
                                         and optimization of short interfering RNA (siRNA) sequences for effective silencing of
                                         IDO expression in cancer cells.



 


		•	Application
                                         of IDO detection and silencing methodology to human DC (in vitro dCellVax).



 


		•	Demonstration
                                         of in vitro augmentation of immunogenicity by IDO-silenced DC as compared to control
                                         DC



 


		o	Mixed
                                         lymphocyte reaction



		o	Cytokine
                                         Production (IL-4, IL-10, IL-12, IFN-gamma)



		o	Increased
                                         Tryptophan, decreased kyneurinine in culture media






Rationale



Although
the main role of the immune system is to protect the body from foreign pathogens, the immune system also requires a “regulatory”
arm in order to prevent immune responses from destroying not only the pathogen, but also the body. Examples of unrestrained immune
responses include autoimmune conditions, in which the immune system starts attacking the pancreas (Type 1 Diabetes), the nervous
system (multiple sclerosis) or collagen protein in the joints (rheumatoid arthritis). A more severe example of unrestrained immune
response is sepsis, where the immune activation against blood borne infections results in death of the patient. The immune system
possesses regulatory cells, whose role in the body is to restrain immunity. One specific type of regulatory cell is the immature
DC. While mature DC are known to be one of the most potent activators of immunity, in the immature state, DC actively suppress
T cell activation. Immature dendritic cells are found in pregnancy to protect the fetus from maternal immune attack against proteins
found on the fetus that are inherited from the father[4]. Immature DC are also
found in tumors, and contribute to tumor escape from the immune system[5]. Other
conditions associated with immune regulation such as viral infections[6], bacterial
infections[7], and remission from autoimmune diseases[8]
are associated with immature DC.


One
of the main molecular mechanisms by which immature DC suppress the immune system is through high expression of the enzyme indolamine
2,3 deoxygenase (IDO). IDO was originally discovered in 1967 in the rabbit intestine and has been the object of renewed attention
by immunologists in view of its capacity to act as an inducible negative regulator of T cell viability, proliferation, and activation
during inflammation. In addition to potential in direct effects by IDO on APC function, IDO has been proposed to suppress T cells
by degrading tryptophan and increasing the level of tryptophan degradation products (kynureneria and quinolinate). Both of these
activities suppress T cell response by inducing T cell apoptosis.


It
has been shown that immune suppression in cancer[9], pregnancy[10],
and viral infection[11] can be overcome by chemical inhibition of IDO. Unfortunately,
chemical inhibitors of IDO, to date, do not possess specificity towards IDO, and are associated with various side effects[12].
Additionally, administration of chemical inhibitors is not selective to immune cells, given that IDO possesses numerous functions,
systemic administration of inhibitors is not likely to induce sufficient suppression of the enzyme where it is needed, in the
tumor associated DC.


Induction
of RNA interference (RNAi) by administration of siRNA represents a highly specific and efficient mechanism of blocking gene expression.
As an endogenous viral defense mechanism, RNA interference (RNAi) uses a specific subset of enzymes that can bind and cleave homologous
transcripts within mammalian and plant cells. Dr. Wei-Ping Min from the University of Western Ontario has previous used siRNA
to silence immune-associated genes for the purpose of inducing immunomodulation in a publication with Dr. David Koos CEO of Regen
BioPharma[13]. Whereas in the mentioned publication the gene for IL-12 was silenced
in DC in order to inhibit immunity, Regen BioPharma believes that silencing of IDO in DC will stimulate cancer immunity.


Goals 


		1.	Establishment
                                         of assay system to detect IDO gene expression in human cancer cell lines



 


Cancer
cell lines such as HeLa (cervical)[14] and MDA-MB-231 (breast)[15]
have been published to express human IDO. RT-PCR methodology methodology with primers specific to human IDO in these
cell lines. This methodology will allow for detection of the IDO gene. It is important to qualitatively establish detection of
this gene so that we can quantify the level of gene silencing that is achieved by the siRNA sequences.


 


		2.	Identification
                                         of novel human siRNA sequences silencing IDO



 


Sequences
will be developed using bioinformatics algorithms, as well as based on prior literature. Approximately 10-20 sequences will be
utilized to identify optimal silencing effect on cancer cell lines. (The use of cancer cell lines is to safe costs on given
optimizing the detection and efficacy of silencing, dendritic cells are not ideal for screening experiments due to difficulty
in expanding them).


 


		3.	Application
                                         of optimized sequences to human DC



 


Once
ideal sequences for detection of IDO and silencing elucidated, they will be applied to human DC. Gene silencing efficacy will
be assessed by RT-PCR and immune stimulating ability will be assessed by ability to induce proliferation of allogeneic lymphocytes.
This will be the human preclinical data that goes into the IND package.


 


Proposed
Protocols


 


Stage
1 of Project: Quantification of IDO mRNA Detection


MDA-MB-231
human breast cancer cells will be obtained from American Type Tissue Culture (ATCC: Manassas, VA) and grown under fully humidified
5% CO2 environment with DMEM supplemented with 10% FBS, 2% sodium pyruvate, non-essential amino acids (2 mM), penicillin (100
units/ml), streptomycin (100 μg/ml), and glutamine (4 mM) (Gibco-BRL). HeLa cells will be grown under the same conditions
with the exception that MEM will be used as the base media. Cells are passaged by trypsinization twice weekly or as needed based
on 75% confluency. For some experiments IDO induction will be achieved by pretreatment with interferon gamma at a concentration
of 1 IU/ml.


Total
RNA will be isolated using the RNeasy Mini Kit (QIAGEN). Specifically, cells will be trypsinized and harvested at a concentration
of 5-10 x 106 cells, as a cell pellet after washing in PBS an appropriate volume of Buffer RLT Plus will be added and the cells
will be vortexted for 30 seconds. This will result in lysis of the cells, with the lysate then being spun at 3 minutes at 15000g.
The supernatant is then removed and applied to a gDNA Eliminator spin column which is then placed in a 2 ml collection tube. Subsequently,
the collected material is centrifuged for 30 s at ≥8000 x g (≥10,000 rpm). The column is discarded and the flow-through
is saved. The same volume (usually 350 μl or 600 μl) of 70% ethanol is added to the flow-through that has been collected.
Up to 700 μl of the sample, including any precipitate, is then added to an RNeasy spin column placed in a 2 ml collection tube
and the tube is centrifuged for 15 s at ≥8000 x g. The flow-through is discarded. 700 μl of Buffer RW1 is then added
to the RNeasy Mini spin column (in a 2 ml collection tube) and centrifuged for 15 s at ≥8000 x g. 500 μl of Buffer
RPE is added to the RNeasy spin column and centrifuged for 15 s at ≥8000 x g. Subsequently 500 μl of Buffer RPE is
added to the RNeasy spin column and centrifuged for 2 min at ≥8000 x g (≥10,000 rpm). The RNeasy spin column will
then be placed in a new 1.5 ml collection tube. Approximately 30–50 μl RNase-free water is added directly to the spin
column membrane and centrifuged for 1 min at ≥8000 x g to elute the RNA. Reverse transcription performed using Moloney
murine leukemia virus reverse transcriptase (Promega) following the manufacturer's instructions. Reverse-transcribed products
will be analyzed on a Mastercyler Ep Realplex (Eppendorf) using the QuantiFast SYBR Green PCR Kit (QIAGEN) according to the manufacturer's
instructions.


As
gene-specific primers, the following oligo-DNAs will be assessed


		a)	Forward
                                         (5′-3′): IDO, GGTCATGGAGATGTCCGTAAGGT



		b)	Forward
                                         5′-GGAAATAGCAGCTGCTTCTGCA-3′; IDO reverse, 5′-CTCCTCAGGGAGACCAGAGCTT3′



		c)	Forward
                                         5′-tgccaaatccacaggaaaat-3′, reverse: 5′-gtttgccaagacacagtctg-3′;



		d)	Forward
                                         5′-caaatccacgatcatgtgaacc-3′, reverse: 5′-agaacccttcatacaccagac-3′,



		e)	Forward
                                         5′-GGAAATAGCAGCTGCTTCTGCA-3′; reverse 5′-CTCCTCAGGGAGACCAGAGCTT-3′



		f)	Forward
                                         5’ TTCAGTGCTTTGACGTCCTG; reverse 5’ TGGAGGAACTGAGCAGCAT 



As
an internal control β-actin mRNA was also amplified using the following primers: β-actin forward, 5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3′;
β-actin reverse, 5′-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3


PCR
products will be size-separated on a 1.5% agarose gel; expression levels
normalized to the GAPDH mRNA product, and will be visualized by SYBR Safe DNA gel staining (Invitrogen).


A
minimum of 3 independent experiments will be performed per sequence assessed. If inducible expression is not observed, sequences
will be modified using internal algorithms, as well as variations in cycle number and annealing/reannealing temperatures.


Anticipated
Results: Sequences from the proposed sequences will possess varying ability to detect mRNA. This will be used as the test system
for human IDO expression. We anticipate significantly higher expression of IDO mRNA in cells that have been pretreated for 48
hours with Interferon Gamma, based on previous publications[16].


Stage
2 of Project: Identification of novel human siRNA sequences silencing IDO.


HeLa
and MDA cells either growing in stable conditions or pretreated with 1 IU/ml of interferon gamma for 48 hours under conditions
identified in stage 1 of the project to induce maximal expression of the IDO gene.


Various
siRNA sequences will be tested that have been previously shown to inhibit IDO or have been generated based on gene-specific algorithms.
To prepare the modified siRNA duplexes, complementary strands were mixed at equal concentrations, then heated at 70° for 1
min and allowed to anneal at 37° for 30 min. Successful annealing will be assessed with polyacrlamide gel electrophoresis.


Specific
sequences tested will include:


sense
strand


	5′-TAATACGACTCACTATAGCCGTGAGTTTGTCCTTTCAA-3′
	5′-TTGAAAGGACAAACTCACGGCTATAGTGAGACGTATTA-3′
	5′-UCACCAAAUCCACGAUCAUUU-3’
	5′-UUUCAGUGUUCUUCGCAUAUU-3’
	5′-GUAUGAAGGGUUCUGGGAAUU-3′
	5′-GAACGGGACACUUUGCUAAUU-3′



DNA
oligonucleotides for the anti-sense strand


	5′-TAATACGACTCACTATAGAAAGGACAAACTCACGGACT-3′
	5′-AGTCCGTGAGTTTGTCCTTTCTATAGTGAGACGTATTA-3′
	5′-PUAUGCGAAGAACACUGAAAUU-3′
	5′-PUAUGCGAAGAACACUGAAAUU-3′
	5′-PUUCCCAGAACCCUUCAUACUU-3′
	5′-PUUAGCAAAGUGUCCCGUUCUU-3′



Cells
will be transfected using the Amaxa Nucleofector Kit V (Amaxa biosystems, Koeln, Germany). Briefly, 3 * 105 cells
were resuspended in 100 μl of the nucleofector solution V and mixed with 1.5 μg of siRNA, then electroporated using
program V005. Alternatively, lipofectamine based transfection may be utilized depending on efficacy.


Suppression
of IDO gene expression will be assessed using the method identified as most sensitive from Project Stage 1.


Anticipated
Results: These experiments will provide ideal siRNA sequences for silencing of the IDO gene. These sequences will be utilized
as the basis of silencing IDO in DC in the next Stage of the project.


Stage
3 of Project: Application of optimized sequences to human DC.


DC
will be generated from peripheral blood of healthy volunteers. Peripheral blood mononuclear cells (PBMC) will be purified from
heparinized blood on lymphoprep cushions (LSM; Organon Teknilra Corp., Rockville, MD) and resuspended in RPMI-10% FCS, and allowed
to adhere to 6-well plates (Costar Corp., Cambridge, MA). After 2 h incubation at 37 Celsius, the nonadherent cells will be removed
and the adherent cells will subsequently be washed in phosphate buffered saline (PBS), followed by detachment by incubation with
Mg 2+ and Ca 2+ free PBS containing 0.5 mM EDTA at 37 Celsius. The adherent fraction is subsequently cultured at 3 x 10(6)/ml
in RPMI-10% FCS supplemented with 50 ng/ml GM-CSF and 1,000 U/ml IL-4. Media is changed every 2 days for a total of 8 days culture.
DC will be isolated by positive selection for CD83 and subsequently exposed to IFN-gamma on day 6 of culture. The siRNA sequences
identified in Stage 2 of the project will be utilized. IDO gene expression will be assessed by RT-PCR using primers identified
in Stage 1 of the project.


Successfully
silenced DC will be assessed for immunological responsiveness by ability to stimulate mixed lymphocyte reaction


Dendritic
Cell Isolation from Human Blood – Adherent Method


Materials:


	Reagent	Vendor	Cat#	Amount	Price
	Recombinant
    Human IL-4 CF	RND
    Systems	204-IL-010/CF	1
    mg	$4,300
    
	 	 	 	10
    μg	$205
    
	 	 	 	50
    μg	$679
    
	Recombinant
    Human GM-CSF	RND
    Systems	215-GM-010/CF	10
    μg	$219
    
	 	 	 	50
    μg	$799
    
	RPMI
    1640 with L glutmine and 25mM HEPES buffer	Life
    Technologies	22400-105	10x
    500ml	$271
    
	RPMI
    1640 with L glutamine	Life
    Technologies	11875-119	10x
    500ml	$188
    
	HEPES
    Buffer	Life
    Technologies	15630-106	20
    ml	$18.50
    
	Beta-mercaptoethanol	Sigma	M3148-25ML	25
    ml	$15.30
    
	6
    well plates	Fisher
    Scientific	08-772-1B	50
    plates	$118.03
    
	50
    ml Centrifuge Tube	Fisher
    Scientific	1443222	500
    tubes	$333.65
    
	Penicillin-Streptomycin
    (100x)	Life
    Technologies	15140-122	100
    ml	$18.91
    
	Human
    Albumin Solution 25%	Gemeni
    Bio Products	800-120	50
    ml	$95
    
	Human
    Serum AB (Heat Inactivated)	Gemeni
    Bio Products	100-512	100
    ml	$159
    
	Trypan
    BLue 0.4%	Sigma	T8154-20ML	20
    ml	$5


 


Reagents
to Prepare:


 


IL-4
Reconstitution:


 


Reconstitute
in PBS with 8% Human Albumin Solution to a final concentration of 20μg/ml.


(Example:
We reconstituted 10μg of IL-4 in 500μL of solution)


 


GM-CSF
Reconstitution:


 


Reconstitute
in PBS with 8% Human Albumin Solution to a final concentration of 20μg/ml.


(Example:
We reconstituted 10μg of IL-4 in 500μL of solution)


 


Dendritic
Cell Culture Media (keep at 37C for culture):


 


RPMI
1640 w/ L-glutamine


25mM
HEPES


1%
Pen Strep


5%
Human Serum AB


50
μM BME (optional)


 


Before
culture make sure to filter with a 0.22μm filter to remove bacterial contaminants


200ml
PBS (1x) – for washing platelets out of supernatant following ficoll separation





Procedure:


		1.	Obtain
                                         10ml of blood sample



		2.	Dilute
                                         1:1 in PBS warmed to RT



		3.	Place
                                         remaining ~180ml PBS on ice



		4.	Underlay
                                         10ml Ficoll



		a.	Slow
                                         down pipette aid to minimize disruptance to interphase layer.



		b.	Be
                                         careful of air bubbles that could disrupt layer



		5.	Carefully
                                         transfer tube into centrifuge being careful not to disrupt interphase layer



		a.	Spin
                                         at 2000RPM, 20min, no brakes @RT



		b.	After
                                         spin is completed set temperature on centrifuge to 4C



		6.	Remove
                                         interphase layer and transfer to new 50ml conical



		a.	Optional
                                         tips: removing the upper layer (containing PRP) for easier access to interphase layer
                                         – this allows you to use a P1000 pipette instead of a pipette aid for greater accuracy



		7.	Add
                                         PBS (on ice or 4C) to solution containing interphase layer to bring the total volume
                                         to 50ml



		8.	Centrifuge
                                         at 1500RPM, 10min, brakes on @4C



		9.	Inspect
                                         clarity of supernatant, repeat wash cycles (900RPM, 10min, no brakes, @4C) until supernatant
                                         is clear (typically 3 wash cycles completed)



		10.	Aspirate
                                         supernatant and resuspend in 1ml DC Culture Media at 4C



		11.	Take
                                         10μL of cell suspension and count using a hemocytometer to determine the number
                                         of wells necessary for culture (approximately 2 million mononuclear cells per well)



		a.	Add
                                         trypan blue to check cell viability



		12.	Plate
                                         6 million cells per well with 3mL warmed DC media per well



		13.	Place
                                         6 well plate in incubator at 37C, 5% CO2 for 1 hour



		14.	After
                                         hour incubation is complete, aspirate supernatant leaving behind adherent cells, wash
                                         4 times with warm PBS



		15.	Replace
                                         with fresh DC Culture Media with 3ml per well



		16.	IL-4
                                         concentration



		a.	Desired
                                         final concentration 10ng/ml



		b.	Therefore
                                         given a stock solution of 20μg/ml, alloquat 3μL per well



		17.	GM-CSF
                                         concentration



		a.	Desired
                                         final concentration 50ng/ml



		b.	Therefore
                                         given a stock solution of 20μg/ml, alloquat 15μL per well



		18.	Place
                                         6 well plate back in incubator at 37C with 5% CO2



		19.	On
                                         day 2 after isolation procedure, replace cytokines



		20.	On
                                         day 4 after isolation procedure, replace cytokines



		21.	On
                                         5th day, add maturation signal for 2 days



		a.	10
                                         ng/ml Lipopolysaccharides for 24 hours



 






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