When the subject is present and both the subject and site staff are ready for administration, products will be formulated in accordance with SOP -UFBTIP- 124 and SOP -UFBTIP-125. Products will be labeled with, at a minimum, the subject’s name and study ID code. Individuals transporting samples must be certified to ship biological products, diagnostic or infectious materials. All packaging must comply with the Department of Transportation (DOT), the Federal Aviation Authority (FAA), and the International Air Transport Association (IATA) dangerous goods regulations. 19.2 TTRNA isolation Surgically resected reMB/PNET specimens are collected aseptically and transferred from the operating room to the research laboratory in accordance with SOP -UFBTIP -116. All specimens obtained at outside hospital facilities will be transferred aseptically under FDA guidelines (BB IND 13630 (Sampson), BB IND 14058 (Mitchell)) in accordance with SOP -UFBTIP -120 or SOP -UFBTIP -130. The tumor is washed of excess blood and minced using sterile scissors. Minced tumor fragments are then snap frozen and stored in liquid nitrogen until RNA extraction. Because tissue thawing in the absence of ribonuclease inhibitors leads to RNA degradation, frozen tumor tissue is homogenized immediately prior to RNA extraction . Total cellular RNA is then extracted using the RNeasy kit (Qiagen, Valencia, CA) following the manufacturer’s specifications. Extracted RNA is quantified with a NanoDrop at 260 nm and subsequent ly used in amplification reactions in accordance with SOP - UFBTIP -102. 19.3 Amplification of RNA For larger samples the total RNA is isolated using RNeasy (Qiagen C at# 74 104). For small tumor samples SuperScript III CellDirect cDNA Synthesis System (Invitrogen) is used to isolate total RNA from the tumor cells. Invitrogen’s CellDirect Resuspension/Lysis Enhancer Buffers (Invitrogen, Cat# 11739- 010) are mixed 10:1 a nd added directly to the autologous tumor cells. The extracted RNA mixture is heated for 10 minutes at 75 OC. An aliquot of the CellDirect lysate or extracted RNA is used for 1st -strand cDNA synthesis with using the primers 5’-AAG CAG TGG TAT CAA CGC AGA GTA CTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTV N -3’; (CDS Primer IIIA), and 5’- AAG CAG TGG TAT CAA CGC AGA GT/G GCC ATA TTG GCC rGrGrG/3AmMC6 -3’(SMART III) at 70OC for 3 minutes, 42OC for 20 Re-MATC H Protocol Page 78 of 106 Version: 20180628 minutes and 70OC for 15 minutes. The 1st -strand cDNA libraries were amplified with ClonTech’s Advantage 2 Polymerase PCR kit (ClonTech Cat# 639201) or ClonTech’s polymerase mix (ClonTech Cat# 639201) by primers (CDS Primer IIIA; and T7 –Smart: 5’ -CCA TCC TAA TAC GAC TCA CTA TAG GTA TCA ACG CAG AGT GGC CAT ATT G- 3’) at 95 OC for 2 minutes, 30- 50 cycles of 95OC for 25 seconds, 65OC for 45 seconds, 68OC for 6 minutes and 68OC for 10 minutes. The integrity of the amplification is evaluated by Real- Time PCR on a 7900HT Fast Real -Time PCR System (Applied Biosystems) or equivalent using gene expression levels of human housekeeping genes (ACTB, EGFR, GAPDH or HPRT). PCR amplifica tion conditions may be modified to optimize amplifications as assessed by housekeeping gene detection. The detection of at least one housekeeping gene and a cDNA smear extended to 1Kb or greater is required to declare the template suitable for RNA synthes is. The amplified cDNA PCR products are purified with QIAquick PCR Purification kit (QIAGEN Inc., Valencia, CA, Cat# 28104). In vitro mRNA transcription was done using the same products and procedures as those previously approved for INDs 12,839, 13,240 a nd 13,630. The amplified mRNA is synthesized using T7 mMessage mMachine™ Transcription Kit (Ambion Inc., Austin, TX, Cat# 1344) from PCR amplified TTRNA cDNA library templates, and cleaned with RNeasy™ kit (QIAGEN, Cat# 74104) and quantified with a NanoDr op at 260 nm. The integrity of the amplified mRNA is verified as defined in the RNA Release Criteria. The RNA amplification process will be conducted in accordance with SOP -UFBTIP -103 through SOP - UFBTIP -115 and released in accordance with SOP -UFBTIP -119 as outlined below. RNA Release Criteria Nucleic acid/protein (260/280) >1.8 Size RNA product range > 1kB by agarose gel electrophoresis. Sterile Negative aerobic and anaerobic bacterial cultures and fungal cultures by Clongen. 19.4 Procedure for TTRNA-DC and TTRNA-xALT Generation Blood will be obtained by leukapheresis and transported to the UF cGMP facility in accordance with SOP - UFBTIP -121 for cells collected at the University of Florida or SOP -UFBTIP- 127 for cells collected at external sites. PBMCs will be isolated in accordance with BR-UFBTIP -001 and plated into flasks for in vitro generation of DCs. Non- adherent cells will be frozen in accordance with BR -UFBTIP -004 and subsequently used for production of antigen -specific T cells. Immature DCs will be generated from the PBMC s in vitro by 7-day culture with GM -CSF and IL -4. At the end of the 7 day incubation, the immature DC s will be harvested and electroporated with TTRNA. The electroporated immature DCs are placed in a flask with AIM V media with HABS containing GM-CSF, IL -4, TNF -α + IL -6 + IL -1β at 37oC, 5% CO 2 for 18 -24 hours for maturation. The cells are washed with PBS and frozen in 80% human AB serum, 10% Cryoserv , and 10% Dextrose in accordance with BR- UFBTIP -002. A portion of t he DCs are frozen and stored in liquid nitrogen until needed for DC vaccination. Another portion of the mature DCs is used for an in vitro 21 day stimulation of antigen -specific T cells in accordance with BR -UFBTIP -003. After freezing and prior to release , an aliquot of cryopreserved cells will be thawed , prepared, and sent to Clongen for QA/QC release testing in accordance with FDA IND BB -14058 and SOP -UFBTIP -122 and SOP -UFBTIP -123. Testing will include sterility ( aerobic and anaerobic bacterial cultures , fungal cultures) , endotoxin, and mycoplasma . A separate aliquot will be prepared for v iability and phenotypic analysis for release criteria in accordance with SOP -UFBTIP -100. For each vaccination, DCs will be rapidly thawed at 37oC, washed with PBS and counted. The DC concentration will be adjusted to 1 x 107 cells per 400 µL of preservative free saline and placed into a sterile tuberculin syringe with a 25 gauge needle in accord ance with SOP -UFBTIP -124. The final thawed and Re-MATC H Protocol Page 79 of 106 Version: 20180628 formulated DC will be stored at 2 -8°C for up to 6 hours. After this time the material will be disposed of if not given to the patient. Autologous ex vivo expanded TTRNA -specific lymphocytes (xALT) Non-adherent cells will be obtained from the initial leukapheresis and frozen in accordance with BR - UFBTIP -004 and will consist of the cells remaining after the plastic adherence step described above for DC generation in accordance with BR -UFBTIP -002. These non-adherent cells are stimulated in vitro for 21 days with mature DCs . 2x107 non-adherent cells are placed into T -25 flasks along with 2x106 matured TTRNA loaded DCs, in 10 mLs AIM V containing 5% HABS and 3,000IU/mL IL -2 and incubated at 37OC for 5 days. After 5 days, the cells will be treated with 100IU/mL IL -2 every 3 days if needed based on observation of cell clusters and P H measurement of media. The cells are expanded into progressively larger flasks as needed. At the end of 21 days, the TTRNA stimulated T cells are harvested, washed in PBS, counted and viably frozen in 90% HABS + 10% Cryoserv in accordance with BR -UFBTIP -003. After freezing and prior to release, an aliquot of cells is thawed and sent for QA/QC release testing according to FDA IND BB -14058 and SOP -UFBTIP -123. This includes aerobic and anaerobic bacterial cultures (1x106 TTRNA -xALT) , fungal cultures (1x106 TTRNA -xALT) , and endotoxin testing . Viability ( >70%) and phenotypic analysis will also be conducted for release criteria in accordance with SOP -UFBTIP -100. In preparation for TTRNA -xALT, cells are rapidly thawed at 37oC, washed with saline + human albumin , and counted. The cell concentration will be adjusted to 3 x 107 cells/ kg (or all cells available if less than target dose) in preservative free saline with 1% human serum albumin and placed into a syringe in accordance with SOP -UFBTIP -125. The final thawed and formulated TTRNA -xALT will be stored at 2 -8°C for up to 6 hours. After this time the material will be disposed of if not given to the patient.