Question 15: Were the cells in the infused product selected / modified / engineered prior to infusion?
Indicate “yes” if the cells contained in the product were selected (i.e. selective retention of a population of desired cells through recognition of specified characteristics), modified or genetically engineered and continue with question 16. Indicate “no” if the cells contained in the product were not selected, modified or genetically engineered in any way prior to infusion and continue with question 40.
Question 16: Specify the portion manipulated:
If the product being infused as a cellular therapy is a portion from a prior HCT (e.g. DLI/DCI), the portion becomes the “entire” product for the purposes of this form. The product can then be further divided.
Indicate the portion of the product that was manipulated. If the entire product was manipulated, select “entire product” and continue with question 18.
If a portion of the product was removed and manipulated, select “portion of product” and continue with question 17.
Question 17: Was the unmanipulated portion of the product also infused?
Indicate “yes” if the unmanipulated portion of the product was also infused. Indicate “no” if the unmanipulated portion of the product was not infused.
Question 18: Was the same manipulation method used on the entire product / all portions of the product?
If the same manipulation was used on the entire product or all portions of the product, indicate “yes”. If different manipulation methods were used indicate “no”. All manipulations for each portion of the product should be reported in questions 19-46.
Question 19-20: Specify method(s) used to manipulate the product: (check all that apply)
Indicate the method(s) of manipulation.
Cultured (ex-vivo expansion): cells were placed in culture to increase in number (i.e. to expand) allowing for sufficient cells for infusion. Continue with question 40.
Induced cell differentiation: cells were placed in culture to give rise to cellular elements with biological characteristics other than those of the cells being cultured (i.e. mesenchymal stromal cells cultured to make osteoblasts; pluripotent stem cells cultured to make neural cell precursors). Usually, the description of the process would include the term “differentiation of cells X into cells Y”. This scenario can be seen in regenerative medicine indications. Continue with question 40.
Cell selection – positive: the manipulation of a cellular therapy product that a specific cell population(s) is enriched. This may be achieved by using an antibody that binds to a specific population of cells (e.g., CD4+ selection). Continue with question 40.
Cell selection – negative: the manipulation of a cellular therapy product such that a specific cell population(s) is reduced. Continue with question 40.
Cell selection based on affinity to a specific antigen: the cellular product undergoes selection to isolate the target population based on the ability of the target population to bind or recognize a specific antigen (e.g. a T cell population recognizing viral proteins, or a protein associated with a cancer).
Continue with question 40.
Genetic manipulation (gene transfer / transduction): cells are manipulated via gene transfer, a process by which copies of a gene are inserted into living cells in order to induce synthesis of the gene’s product; or transduction, a process by which foreign DNA is introduced into a cell by a virus or viral vector. These techniques deliberately alter the genetic material of an organism in order to make them capable of making new substances or performing new or different functions. Continue with question 21 to report the types of genetic manipulation.
Other cell manipulation: not fitting an above category. Specify manipulation in question 20 and continue with question 40.
Question 21-29: Transfection:
Transfection is a process of deliberately introducing naked or purified nucleic acids by viral or non-viral methods into eukaryotic cells. If the product underwent transfection, continue with question 22. Else continue with question 30.
Viral transduction: Viral transduction is a process by which nucleic acid (DNA) is introduced into a cell by a virus, followed by viral replication in the affected cell. If “yes”, indicate the virus used in the viral transduction in questions 23 and 24. Indicate “no” if the product did not undergo viral transduction and continue with question 25.
: Lentiviruses are members of the genus of retroviruses that have long incubation periods and cause chronic, progressive, usually fatal disease in humans and other animals. Indicate “no” if a Lentivirus was not used for the viral transduction.
: Retroviruses are any group of RNA viruses that insert a DNA copy of their genome into the host cell to replicate. HIV is an example of a Retrovirus. Indicate “no” if a Retrovirus was not used for the viral transduction.
Non-Viral transfection: Non-viral transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. If “yes”, indicate the method of non-viral transfection in question 26-29. Indicate “no” if the product did not undergo non-viral transfection and continue with question 30.
: Transposons are discrete mobile sequences in the genome that can transport themselves directly from one part of the genome to another without the use of a vehicle such as phage or plasmid DNA. They are able to move by making DNA copies of their RNA transcripts which are then incorporated into the genome at a new site. Indicate “no” if Transposons were not used for the non-viral transfection.
: Electroporation is a process of introducing DNA or chromosomes into cells using a pulse of
electricity to briefly open the pores in the cell membranes. Indicate “no” if Electroporation was not used for the non-viral transfection.
: Indicate “yes” if a different non-viral transfection method not previously listed was utilized. Specify the other non-viral transfection method in question 29.
Question 30-32: Gene editing:
Gene editing is a type of genetic engineering in which DNA is inserted or removed from a genome using artificially engineered nucleases. If “yes”, specify which gene was edited in the manipulation in question 31.
If “other gene” is answered for question 31, specify the gene in question 32. Indicate “no” for question 30 if the cells did not undergo gene editing.
Question 33: Were cells engineered to express a non-native antigen receptor?
Indicate “yes” if the cells underwent a type of genetic engineering in which a gene is transferred codes for an antigen receptor other than one that may already be naturally present in the cell (e.g. T-cells have natural T-cell receptors [TCRs]; a transgenic TCR or a Chimeric Antigen Receptor [CAR] are non- native antigen receptors). Indicate “no” if the cells did not undergo transfer of such a gene and continue with question 38.
Question 34-37: Specify the protein inserted into the cellular product:
Specify which construct was utilized as part of the genetic manipulation process:
T-cell receptor: Heterodimeric antigen receptors present on the surface of T-cells. Continue with question 38.
Chimeric Antigen Receptor (CAR): A cell-surface receptor that has been engineered to combine novel features and specificities from various sources in order to enhance its antigen specificity. Engineered T- cells or B-cells will produce the specialized receptor that will be capable of binding to an epitope on its target cell1.
The CAR construct consists of several genes that can exert different functions, such as augment the immune response by co-stimulation, increase affinity, and increase the time it persists in the circulation without being cleared. The CAR construct information is usually unique and may influence its effect against the disease or the severity of side effects. Specify which construct(s) was used in the making of the Chimeric Antigen Receptor (CAR) in question 35. If a construct was utilized that is not in the list, check “other construct” and specify in question 36.
For more information related to the different constructs and their functions, see this article: https://www.jci.org/articles/view/80010.
Suicide gene: Cells underwent manipulation to have cell suicide inducing transgenes inserted into the product. Specify the suicide gene in question 37.
Question 38-39: Other genetic manipulation:
Indicate “yes” for other genetic manipulation that does not fit into a category listed above and specify in question 39.
Question 40: Was the product manipulated to recognize a specific target/antigen?
Indicate “yes” if the cells were cultured or engineered so that the majority of cells in the end product are able to recognize or bind to a chosen target (e.g. proteins from a virus or a protein from a tumor) and continue with question 41. This manipulation can be done outside of the context of ‘genetic manipulation’. If “no”, continue with question 47.
Question 41: Specify viral target(s): (check all that apply):
If the target is viral, continue with question 42.
If the target is tumor/cancer antigen, continue with question 44.
If the target is something other than viral or tumor/cancer antigen, continue with question 46.
Question 42-43: Specify the target antigen: (check all that apply)
Select all target viral antigen(s) that apply to the product. If the target is “other virus”, specify in question
43. Continue with question 47.
Question 44-45: Specify the tumor / cancer antigen: (check all that apply)
Select all target tumor/cancer antigen(s) that apply to the product. If the target is “other tumor/cancer antigen”, specify in question 45. Continue with question 47.
Question 46: Specify other target:
If the target is something other than viral or tumor/cancer antigen as selected in question 41, specify the other target. Continue with question 47.
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