High-Throughput HLA-Typing: on Raw, Unpurified Cord Blood Samples

描述(申请人提供)：摘要。未被满足的机会：准确、简单、廉价、高通量的人类白细胞抗原配型，作为支持国家规模干细胞治疗计划的伴随性基因测试。很明显，出生时即可从脐带(UCB)获取的50-100毫升血液可以成为一种丰富、相对廉价的多潜能间充质干细胞来源，用于治疗淋巴样癌、地中海贫血以及最近作为潜在干细胞疗法的更大范围的潜在来源。随着正在进行的研究揭示了脐带血干细胞疗法的全部内容(糖尿病、神经疾病、心血管疾病、创伤等)，将脐带血干细胞的收集和处理扩大到接近出生队列的水平是可行的(在美国为12,000/天)。然而，为了支持即使是10%应计比率，也需要大大加强联合商业委员会的样本处理和样本分析。在这份R21中，我们专注于这种异基因脐带血干细胞治疗所需的伴随性遗传测试的放大：对处理后的脐带血干细胞样本和将从中受益的患者群体进行HL A分型。解决方案：廉价的微阵列，用于新鲜脐带血的原始干细胞的人类白细胞抗原分型，或者更好的是，从脐带血中收集并在格思里卡片上干燥运输。我们将优化和验证一套简单而廉价的用于人类白细胞抗原分型的微阵列技术，我们已经在初步结果中展示了这一技术，用于处理原始的、完全未经净化的成人血液和匹配的原始样本，这些样本收集并在Gucere卡上干燥运输。对于这个R21来说，特别重要的是可以从NHLBI资助的COBLT研究中获得高度验证的(原始)UCB样本，这是一个开创性的UCB c0nsortium(见支持函)。COBLT已经利用“金标准”基因分型技术，在这些脐带血样本上获得了高分辨率的人类白细胞抗原分型。在这本R21中，我们将重点介绍已知与UCB治疗结果有最强相关性的HLA-A、HLA-B和DRB1基因，以及已有初步数据表明其在优化UCBT结果中的作用的HLA-C和DQB1。具有特殊后勤重要性的是我们的演示，即通过一种新的回收方法，我们只需添加水就可以生产出准备好分析的样本，从而优化这种“原始样本人类白细胞抗原配型”，以对干燥的浓缩脐带血样本进行工作，收集并以格思里卡片运输。验证这套支持基于脐带血的干细胞治疗的耦合微阵列技术将得到4个具体目标的支持：SA1。年1、2、制造低成本的人类白细胞抗原芯片[人类白细胞抗原A、B、C、DRB1、DQB1]。SA2.YR-1。优化480份原始的、浓缩的脐带血样本的人类白细胞抗原分型。SA3.YR-2。优化480份未加工、浓缩的脐带血标本的人类白细胞抗原分型：在格思里卡片上干燥。SA4.YR-2。对未加工的、浓缩的脐带血样本进行自动的人类白细胞抗原分型：新鲜和干燥。与后续的R33的关系。在此R21中，我们将演示5uL浓缩的UCB可以通过管道输送到Guslee Card，邮寄干燥，然后通过“只加水”进入我们的高通量HLA配型工作流程。在后续的R33中，我们将利用R21验证作为更大规模的前瞻性验证的基础，与NHLBI附属组织进行验证，例如世界骨髓捐赠者协会的脐带血工作组。尽管雄心勃勃，但这一想法是可行的：考虑到这一R21团队(BK)中的一人曾担任NMDP国际委员会主席。在R33中，目前用于收集、处理和人类白细胞抗原配型标准操作程序将继续不变：但同时，每个丰富的脐带血样本中的5uL将在加工点被转移到格思里卡，匿名，在国际上邮寄到Genusa，用于高通量的人类白细胞抗原配型，在R21开发的系统的放大版本上。然后，在准确度和成本方面与目前对纯化DNA进行的人类白细胞抗原分型方法进行比较。

DESCRIPTION (provided by applicant): Abstract. The Unmet Opportunity: Accurate, simple, inexpensive, high throughput HLA-Typing, as the Companion Genetic Test to support a national-scale program of stem cell therapy. It has become clear that the 50-100mls of blood, readily obtained at birth from the umbilical cord (UCB) can become an abundant, relatively-inexpensive source of pluripotent mesenchymal stem cells to be used in the treatment of lymphoid cancers, the thalassemias and more recently, as the potential source for a much larger range of potential stem cell therapies. As ongoing research reveals the full pallet of cord blood stem cell therapies (diabetes, neurological disease, cardiovascular disease, trauma, etc) it could be feasible to scale-up the collection and processing of UCB stem cells to a level that would approximate the birth cohort (@12,000/day in the US). However, to support even 10% of that accrual rate, substantial enhancement of UCB sample processing and sample analysis will be required. In this R21, we focus on the scale-up of the Companion Genetic Test required for such allogenic UCB stem cell therapy: HLA-Typing of both the processed UCB stem cell samples and the patient population who would benefit from them. The Solution: Inexpensive Microarrays for HLA-Typing of raw stem cells from fresh umbilical cord blood, or better, from UCB collected and shipped dry on Guthrie Cards. We will optimize & validate a simple & inexpensive suite of microarray technologies for HLA-Typing, which we have already shown in Preliminary Results, to work on raw, completely-unpurified adult blood and on matched raw samples collected & shipped dry on Guthrie Cards. Of special importance to this R21, is access to highly-validated (raw) UCB specimens from the NHLBI-funded COBLT study, the seminal UCB c0nsortium (see Letter of Support). High resolution HLA-typing has already been obtained, by COBLT, on those UCB samples, using "gold-standard" genotyping technologies. In this R21, we will focus on HLA-A, HLA-B and DRB1 genes, which are known to have the strongest associations with UCB therapeutic outcome and HLA-C & DQB1 for which there are preliminary data to suggest a role in optimized UCBT outcomes. Of special logistical importance will be our demonstration that such "Raw Sample HLA-Typing" can be optimized to work on dried enriched UCB samples, collected and shipped on Guthrie cards, via a novel recovery approach where we "just add water" to produce a sample ready for analysis. Validation of this set of coupled microarray technologies, in support of UCB-based stem cell therapy, will be supported by 4 Specific Aims: SA1. Yrs 1,2. Manufacture low-cost HLA-Chips [HLA-A, HLA-B, HLA-C, DRB1 & DQB1]. SA2. Yr-1. Optimize HLA-Typing with 480 raw, enriched UCB samples. SA3. Yr-2. Optimize HLA-Typing on 480 raw, enriched UCB specimens: dry on Guthrie Cards. SA4. Yr-2. Automated, HLA-Typing of raw, enriched UCB samples: fresh & dried. Relation to a Follow-on R33. In this R21, we will demonstrate that 5uL of enriched UCB can be pipetted to a Guthrie Card, mailed dry, then fed-into our high throughput HLA-Typing workflow by "just adding water". In a follow-on R33, we will leverage this R21 validation as the basis for a much larger, prospective validation with a NHLBI-affiliated group such as the Cord Blood Working Group of the World Marrow Donor Association. Although ambitious, that idea is feasible: given that one of this R21 Team (BK) was previously Chairman of the International Committee of The NMDP. In that R33, the current SOPs for collection, processing & HLA-Typing would continue unaltered: but, in parallel, 5uL of each enriched UCB sample would be transferred to a Guthrie Card at point of processing, anonymized, mailed internationally to GenUSA for high throughput HLA-Typing, on a scaled-up version of the system developed in the R21. That "Raw Guthrie Card" HLA-Typing would then be compared, in terms of accuracy & cost, to that obtained by current methods of HLA-Typing on purified DNA.