**5. Cure of nonmalignant diseases by cord blood transplantation**

In **Table 2**, to assess whether this observation of the superior efficacy of MaxCell CB can be extended to other nonmalignant diseases, Chow's group conducted a multicenter study of transplantation of 120 patients who received MaxCell CB for nonmalignant diseases—46 for thalassemia, 3 for sickle cell disease and 71 other nonmalignant indications [5]. To maximize cell dose, besides using exclusively MaxCell CB, double CBT was used in 12% of the cases and no-wash thaw procedure was used in 58% of the patients. Median TNC dose was 10.5 × 107 /kg at collection and 7.7 × 107 /kg on infusion, and median CD34+ cell dose was 3.7 × 105 /kg at collection. Twenty-six, forty-eight and fifty-three patients were matched at zero, one and two or more mismatches at HLA A/B/DR loci. Myeloid ANC500 and platelet 20K engraftment occurred at a median of days +21 and +49, respectively. The cumulative incidences to myeloid and platelet 20K engraftment were 87 ± 6% and 81 ± 6%, respectively. Autologous recovery occurred in only 3 ± 2% of the patients in this population made up of 38% thalassemics. Importantly, OS at 3-year was 79 ± 4%, whereas 3-year DFS was 70 ± 6%, respectively, with 100 days and 3-year TRM at 10 ± 3% and 20 ± 4%, respectively. Within the statistical power of this series, univariate analysis showed that ABO match, recipient sex, age, myeloablative conditioning regimen and CMV seropositivity were nonsignificant predictors of particular outcome. Double CBT was associated with a significantly higher incidence of acute GvHD grades II–IV (relative risk 2.23; p = 0.05). Higher pre-freeze CD34+ dose improved myeloid (RR = 1.55; p = 0.05) and platelet (RR = 2.73; p = 0.05) engraftment, OS (RR of death = 0.30; p =  0.05) and DFS (RR of death or relapse = 0.27; p = 0.02). In this study, TNC was not a significant factor, unlike previous reports [3], probably because the usage of typical TNC dose thresholds of 2.5 or 4.0 × 107 /kg for analysis as in the Ruggeri et al. series was not applicable in this series, due to the low numbers of MaxCell CBT patients with such low TNC doses, making statistical comparisons impossible. This anomaly is due to the significantly higher median and average TNC doses afforded by the usage of MaxCell CB products.

By using these three simple strategies to improve infused cell dose—exclusive MaxCell CB usage, not washing cord blood upon thawing (58%) and double CBT (12%) in this series at 46 U.S. and international centers, with divergent nonmalignant diseases, conditioning and GvHD prophylaxis regimens—results consistently superior to other reported series using unrelated RCR CB were obtained as shown in **Table 2** [3, 68]. In fact, these results approached that of Jaing et al. [6] in 35 thalassemia patients in a controlled environment at a single institution and also transplanted exclusively with unrelated donor MaxCell CB that were 100% directly infused upon thawing, proving that the adoption of this combination approach may be efficacious in diverse nonmalignant settings, such as HIV infection and autoimmune diseases.

## **6. Cure of HIV infection by cord blood transplantation**

The application of the highly active antiretroviral therapy (HAART) has significantly improved the survival of HIV-infected patient and converted HIV infection into a chronic but mostly nonlethal disease for those patients who can afford and tolerate HAART in developed countries. Though significantly improving HIV treatment and patient survival, HAART alone is not sufficient to remove the virus in the long term, with rebound expected without continuous HAART treatment due to the long half-life of latent infected cells [72]. HAART cannot cure patients of HIV infection as clinically undetectable plasma viremia may only be achieved by life-long treatment with serious side effects and risks of viral rebound whenever treatment is interrupted. The reservoir of latent HIV provirus persists in patients' latent infected CD4+ T cells even with continued HAART and remains the major obstacle in achieving functional cure for HIV despite the countless efforts to eliminate it. Compared with HIV-negative people, HIV-infected patients are more prone to hematological malignancies including Hodgkin disease (HD) and non-Hodgkin lymphoma (NHL) [73]. Hence, to optimize the life expectancy and quality of life, and to reduce the economic burden of patients, actual cure of HIV is always preferable. Abbreviated life expectancy, high costs, serious chronic side effects and patient noncompliance of HAART therapy drives the search for a HIV cure. Due to existing techniques in leukemia treatment, HSCT has been investigated as a favorable approach to eliminate the HIV virus reservoir while also curing concomitant malignant diseases in the same patient.

The first clinical attempt to use allogeneic HSCT for cure of HIV infection was performed by Hassett et al. [74]. The patient did not improve clinically, and the immunological status remained stable or worsened. Retrospective analysis of reported cases by Hütter and Zaia [75] showed negligible differences between HIV-positive and HIV-negative patients following allogeneic HSCT. Such results are to be expected as HIV produced by the latently infected host cells that survive the allogeneic HSCT re-infects the newly engrafted donor immune system. HAART administration during and following allogeneic HSCT did not change the clinical course, and HIV in all eight patients in these seven reports who discontinued HAART after HSCT rebounded in just a couple of days [76]. Of these, two patients from the Brigham & Women's Hospital were reported initially to be negative for HIV using the most sensitive techniques available following unrelated donor 10/12 HLA-matched HSCT. Unfortunately, after HAART suspension, the virus rebounded in both patients within a few weeks. Without the continued antiretroviral treatment, patients show viral rebound quickly and thus were not functionally cured of HIV. In the study by Henrich et al., two patients received allogeneic HSCT from wild-type chemokine receptor 5 (CCR5) donors and were both reported to be free of detectable virus [77]. However, when HAART treatment was interrupted, viral RNA and proviral DNA became positive again 12 and 32 weeks later [78]. Taken together, these attempts appear to indicate that allogeneic HSCT alone is insufficient to eradicate HIV-1 infection.

due to the low numbers of MaxCell CBT patients with such low TNC doses, making statistical comparisons impossible. This anomaly is due to the significantly higher median and average

By using these three simple strategies to improve infused cell dose—exclusive MaxCell CB usage, not washing cord blood upon thawing (58%) and double CBT (12%) in this series at 46 U.S. and international centers, with divergent nonmalignant diseases, conditioning and GvHD prophylaxis regimens—results consistently superior to other reported series using unrelated RCR CB were obtained as shown in **Table 2** [3, 68]. In fact, these results approached that of Jaing et al. [6] in 35 thalassemia patients in a controlled environment at a single institution and also transplanted exclusively with unrelated donor MaxCell CB that were 100% directly infused upon thawing, proving that the adoption of this combination approach may be efficacious in diverse nonmalignant settings, such as HIV infection and autoimmune diseases.

The application of the highly active antiretroviral therapy (HAART) has significantly improved the survival of HIV-infected patient and converted HIV infection into a chronic but mostly nonlethal disease for those patients who can afford and tolerate HAART in developed countries. Though significantly improving HIV treatment and patient survival, HAART alone is not sufficient to remove the virus in the long term, with rebound expected without continuous HAART treatment due to the long half-life of latent infected cells [72]. HAART cannot cure patients of HIV infection as clinically undetectable plasma viremia may only be achieved by life-long treatment with serious side effects and risks of viral rebound whenever treatment is interrupted. The reservoir of latent HIV provirus persists in patients' latent infected CD4+ T cells even with continued HAART and remains the major obstacle in achieving functional cure for HIV despite the countless efforts to eliminate it. Compared with HIV-negative people, HIV-infected patients are more prone to hematological malignancies including Hodgkin disease (HD) and non-Hodgkin lymphoma (NHL) [73]. Hence, to optimize the life expectancy and quality of life, and to reduce the economic burden of patients, actual cure of HIV is always preferable. Abbreviated life expectancy, high costs, serious chronic side effects and patient noncompliance of HAART therapy drives the search for a HIV cure. Due to existing techniques in leukemia treatment, HSCT has been investigated as a favorable approach to eliminate the HIV virus reservoir while also curing concomitant malignant diseases in the same patient.

The first clinical attempt to use allogeneic HSCT for cure of HIV infection was performed by Hassett et al. [74]. The patient did not improve clinically, and the immunological status remained stable or worsened. Retrospective analysis of reported cases by Hütter and Zaia [75] showed negligible differences between HIV-positive and HIV-negative patients following allogeneic HSCT. Such results are to be expected as HIV produced by the latently infected host cells that survive the allogeneic HSCT re-infects the newly engrafted donor immune system. HAART administration during and following allogeneic HSCT did not change the clinical course, and HIV in all eight patients in these seven reports who discontinued HAART after

TNC doses afforded by the usage of MaxCell CB products.

196 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

**6. Cure of HIV infection by cord blood transplantation**

As CCR5 is a required co-receptor with CD4 for entry of HIV-1 CCR5-tropic strains, and the mutated form of CCR5 with a 32bp deletion (CCR5-Δ32 mutation) provides resistance to the CCR5-tropic strains of HIV-1 in people homozygous for such mutation [79], Chow reasoned that HSCT with CCR5-Δ32/Δ32 donor will confer such resistance to recipients after developing complete donor chimerism, and if such recipients happen to be HIV-infected and contain only or predominantly CCR5-tropic viruses, such patients may be cured of their HIV infection. In 2001, Chow et al. were the first to propose the use of CCR5-Δ32/Δ32 donor HSCT to cure HIV infection and went on to file a patent application on the concept [80]. In vitro, CD4 cells from people homozygous for CCR5-Δ32 are highly resistant to infection by CCR5-tropic HIV-1, the dominant strains in vivo [81]. Even HIV-infected patients with heterozygous CCR5-Δ32 mutation genotype appear to derive partial benefits in the form of slower progression to acquired immunodeficiency syndrome status [82, 83].

Chow's approach was subsequently validated by Hütter et al. several years later [84]. This HIVinfected patient, now known commonly as the "Berlin patient," became the first and only known case in which a HIV-infected patient was functionally cured of HIV infection and survived [85]. Dr. Hütter later acknowledged that the technology first disclosed by Chow et al. in their 2001 U.S. patent application #*09/998,832* [80] and subsequently in U.S. patent application *2003/0099621 AI* was the basis for the Berlin patient cure [10, 86–88], when he wrote "…in 2001, R. Chow, founder of StemCyte, Inc., applied for a patent to screen allogeneic stem cell donors for a beneficial gene with which to treat HIV infection (U.S. patent *2003/0099621* AI)." [88]. The Berlin patient was diagnosed with acute myeloid leukemia and HIV infection and received an initial HSCT from an adult donor homozygous for the CCR5-Δ32 mutation. The patient's leukemia subsequently relapsed, prompting a re-transplantation with graft from the same donor. To this date, since the first transplantation, the patient has been free of HIV-1 infection by viral RNA load or proviral DNA in peripheral blood, gut, liver and brain tissue samples, even though HAART was stopped during and after the first HSCT. The Berlin patient achieved complete chimerism with homozygous CCR5-Δ32 genotype in his peripheral blood monocytes after initial transplantation, and subsequently, after re-transplantation with the same donor after leukemia relapse. With the absence of antiretroviral treatment for more than 8 years, viral RNA or proviral DNA has been undetectable in various tissues even with the most sensitive techniques and has shown a complete clinical remission of HIV infection [76, 86]. Although the case of the Berlin patient sheds light on one approach of curing HIV infection, this result has not been easy to replicate on a large scale to date.

To expand on the success of the Berlin patient, Petz and Chow reasoned that the odds of finding a 10/12 HLA A/B/C/DR/DP/DQ-matched adult donor who is also homozygous for the CCR5- Δ32 genotype is several logs more difficult than the probability of a 4/12 HLA A/B/C/DR/DP/ DQ (matched for HLA A/B/DR loci only)-matched donor CB with the CCR5-Δ32/Δ32 genotype [87, 89, 90]. Starting in 2001, in collaboration with John Zaia, Joseph Rosenthal, John Rossi and Stephen Forman of City of Hope, Chow and Petz started screening StemCyte's CB inventory for the homozygous CCR5-Δ32/Δ32 and heterozygous CCR5-Δ32 genotypes. As Hütter et al. commented in 2011 "…Chows' group built a database with over 10,000 cord blood units, genotyped for the CCR5-delta32 deletion [10]." By 2007, Chow, Petz and City of Hope investigators started to plan for a clinical trial with Yvonne Bryson, Ron Mitsuyasu, Mary Territo, Ted Moore and Tempe Chen from UCLA [90]. By 2009/2010, the CB inventory screening process was expanded to include additional CB banks, and probability of finding matched CB units at various HLA match level and cell doses was calculated with the assistance of the National Marrow Donor Program [87]. By 2013, 180 CCR5-Δ32/Δ32 CB units have been identified [89].

To demonstrate the feasibility of CCR5-Δ32/Δ32 CBT to prevent HIV infection in an in vitro model, Petz et al. reported a case when a HIV-negative adult with acute myelogenous leukemia received CCR5-Δ32/Δ32 CBT [87], who engrafted showing complete donor chimerism. In vitro HIV infectivity tests were performed using the patient's engrafted CCR5-Δ32/Δ32 peripheral blood mononuclear cells (PBMCs) challenged with both CCR5-tropic and CXCR4-tropic HIV laboratory strains (BAL and NL4-3). Both viral strains showed no replication activities when cultured with the patient's engrafted CCR5-Δ32/Δ32 PBMCs, but exhibited robust replication with PBMC from control wild-type (WT) CCR5 or heterozygote CCR5/CCR5-Δ32 individuals, thus demonstrating resistance to HIV-1 of CBT derived donor PBMCs. These in vitro results further support the feasibility of curing HIV by CCR5-Δ32/Δ32 CBT.

**Table 4** highlights the eight cases of using homozygous CCR5-Δ32/Δ32 donor HSCT performed up to 2014, with five using adult donors and three using CB plus or minus a second graft. Except for the two patients performed in Berlin (the Berlin patient and one patient with yet unpublished outcome), all have expired [91]. In the three adult donor cases performed outside of Berlin, one died from infection after 4 months and one from pneumonia shortly posttransplantation. In the last case, CXCR4-tropic virus rebounded after transplantation [92] and the patient expired from Non-Hodgkin's Lymphoma relapse after 12 months. Prior to transplantation, the patient appeared to have mixtures of CCR5-tropic and CXCR4-tropic viruses, though HAART therapy prior to transplantation caused a shift to predominantly CCR5-tropic viruses. CXCR4-tropic viruses emerged as the dominant viral type after HSCT probably as a result of the selection in the absence of functional CCR5 receptor. Though rebound occurred with HIV with alternative tropism, this case confirms the effectiveness of the CCR5-Δ32/Δ32 blockade of CCR5-tropic virus cell entry. Moreover, the emergence of viruses using alternative co-receptors does not negate the possibility of a sterilization cure using this approach of patients infected predominantly with CCR5-tropic HIV, since it was the only case out of seven with known outcome where viral tropism switch occurred.

86]. Although the case of the Berlin patient sheds light on one approach of curing HIV infection,

To expand on the success of the Berlin patient, Petz and Chow reasoned that the odds of finding a 10/12 HLA A/B/C/DR/DP/DQ-matched adult donor who is also homozygous for the CCR5- Δ32 genotype is several logs more difficult than the probability of a 4/12 HLA A/B/C/DR/DP/ DQ (matched for HLA A/B/DR loci only)-matched donor CB with the CCR5-Δ32/Δ32 genotype [87, 89, 90]. Starting in 2001, in collaboration with John Zaia, Joseph Rosenthal, John Rossi and Stephen Forman of City of Hope, Chow and Petz started screening StemCyte's CB inventory for the homozygous CCR5-Δ32/Δ32 and heterozygous CCR5-Δ32 genotypes. As Hütter et al. commented in 2011 "…Chows' group built a database with over 10,000 cord blood units, genotyped for the CCR5-delta32 deletion [10]." By 2007, Chow, Petz and City of Hope investigators started to plan for a clinical trial with Yvonne Bryson, Ron Mitsuyasu, Mary Territo, Ted Moore and Tempe Chen from UCLA [90]. By 2009/2010, the CB inventory screening process was expanded to include additional CB banks, and probability of finding matched CB units at various HLA match level and cell doses was calculated with the assistance of the National Marrow Donor Program [87]. By 2013, 180 CCR5-Δ32/Δ32 CB units have been

To demonstrate the feasibility of CCR5-Δ32/Δ32 CBT to prevent HIV infection in an in vitro model, Petz et al. reported a case when a HIV-negative adult with acute myelogenous leukemia received CCR5-Δ32/Δ32 CBT [87], who engrafted showing complete donor chimerism. In vitro HIV infectivity tests were performed using the patient's engrafted CCR5-Δ32/Δ32 peripheral blood mononuclear cells (PBMCs) challenged with both CCR5-tropic and CXCR4-tropic HIV laboratory strains (BAL and NL4-3). Both viral strains showed no replication activities when cultured with the patient's engrafted CCR5-Δ32/Δ32 PBMCs, but exhibited robust replication with PBMC from control wild-type (WT) CCR5 or heterozygote CCR5/CCR5-Δ32 individuals, thus demonstrating resistance to HIV-1 of CBT derived donor PBMCs. These in vitro results

**Table 4** highlights the eight cases of using homozygous CCR5-Δ32/Δ32 donor HSCT performed up to 2014, with five using adult donors and three using CB plus or minus a second graft. Except for the two patients performed in Berlin (the Berlin patient and one patient with yet unpublished outcome), all have expired [91]. In the three adult donor cases performed outside of Berlin, one died from infection after 4 months and one from pneumonia shortly posttransplantation. In the last case, CXCR4-tropic virus rebounded after transplantation [92] and the patient expired from Non-Hodgkin's Lymphoma relapse after 12 months. Prior to transplantation, the patient appeared to have mixtures of CCR5-tropic and CXCR4-tropic viruses, though HAART therapy prior to transplantation caused a shift to predominantly CCR5-tropic viruses. CXCR4-tropic viruses emerged as the dominant viral type after HSCT probably as a result of the selection in the absence of functional CCR5 receptor. Though rebound occurred with HIV with alternative tropism, this case confirms the effectiveness of the CCR5-Δ32/Δ32 blockade of CCR5-tropic virus cell entry. Moreover, the emergence of viruses using alternative co-receptors does not negate the possibility of a sterilization cure using this approach of

further support the feasibility of curing HIV by CCR5-Δ32/Δ32 CBT.

this result has not been easy to replicate on a large scale to date.

198 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

identified [89].


**CB** = cord blood; **AML** = acute myelogenous leukemia; **MDS** = myelodysplastic syndrome; **NHL** = non-Hodgkin's lymphoma; **ALL** = acute lymphoblastic leukemia; **ART** = anti-retroviral therapy; **GvHD** = graft-versus-host disease. Note: One more CCR5-Δ32/Δ32 Donor HSCT was performed on a non-HIV-infected patient [87].

**Table 4.** CCR5-Δ32/Δ32 donor HSCT performed on HIV-infected patients up to 2014 [10, 75–76, 84–96].

In the three cases where CBT was employed with a CCR5-Δ32/Δ32 CB graft, one was a single CBT, with this Minneapolis patient expiring from GvHD after 3 months [91]. In the other two cases (the Barcelona and Dutch patients), both patients died from disease relapse prior to the fourth month, with the Dutch patient also suffering from pneumonia [91, 93, 94].

The Barcelona case was a 37-year-old HIV-1-infected patient with aggressive lymphoma who failed after five rounds of radiochemotherapy and an autologous HSCT. In the absence of a matched sibling donor, the patient received an allogeneic 4/6 HLA A/B/DR-matched unrelated donor CCR5-Δ32/Δ32 CB product from the StemCyte inventory, supplemented with purified CD34+ cells from a haploidentical sibling [95, 96]. By genotypic and phenotypic analysis, the Barcelona patient's HIV strain was CCR5-tropic, with 2.1 copies of replication competent HIV per 107 CD4 T cells and 303 copies per ml enumerated by single copy assay. After HSCT with CCR5-Δ32/Δ32 CB and haploidentical sibling donor purified CD34+ cells, plasma HIV DNA load was confirmed to be undetectable using ultrasensitive analysis. Upon reaching full chimerism with the CB donor, the patient's engrafted CCR5-Δ32/Δ32 CD4 T cells responded to proliferation and activation stimuli in vitro but was resistant to infection by the patient's viral isolate and laboratory strains. Unfortunately, death related to lymphoma progression prevented long-term monitoring of the patient's status; however, this case shows the potential promise and utility of CCR5-Δ32/Δ32 CBT in curing HIV if the patient was infected with CCR5 tropic virus [96].

Despite incredible worldwide attention and many attempts to replicate the success of the Berlin patient, why has there been no other successful cured long-term survivors reported to date? For one, HSCT is a highly risky procedure, with expected worse survival in advanced cases of malignancies. Malignancy is a feature which all of these seven cases share [91]. Four of the six cases expired because of or partially due to relapses from their malignant conditions. The other cases died from infection or pneumonia—all common causes of post-transplant mortalities. Only one case out of eight reported viral rebound, albeit with virus of a different tropism which already existed in the patient prior to transplantation, perhaps indicating that viral rebound through tropism switch is not as easy as one might hypothesize [92].

Currently, the most important barrier to having a large CCR5-Δ32/Δ32 adult donor or CB inventory is the expense and logistical difficulties of screening millions of adult donors or hundreds of thousands of CB archival samples. The 26,000 StemCyte CB units, 10,000 CB units from the M.D. Anderson bank and 8000 adult donors tested in the German Red Cross Donor Registry represent the largest repositories screened for CCR5-Δ32/Δ32 genotypes to date. As such, the current availability of HLA-matched CCR5-Δ32/Δ32 homozygous donors is still extremely limited. The frequency of homozygous CCR5-Δ32 is around 1% in Caucasians, even less in the Middle East and 0% or almost 0 in Africa and in Asian countries like Taiwan, China and Japan [75, 87]. The requirement for rigorous co-selection of both suitable HLA matches and CCR5-Δ32/Δ32 genotypes leads to difficulties in finding appropriate adult donors to treat HIV infection with hematopoietic stem cells from adult donors. Cord blood, however, can tolerate 1 or 2 HLA A/B/DR mismatches and does not necessarily need HLA matches for HLA C/DP/DQ loci; therefore, a CCR5-Δ32/Δ32 CB inventory gives rise to a higher probability of finding a suitable CCR5-Δ32/Δ32 CB unit for individual patients [87, 97]. Instead of needing to find a 10/12 HLA A/B/C/DR/DP/DQ match for homozygous CCR5-Δ32/Δ32 adult donors, the chances of a 4/12 A/B/C/DR/DP/DQ homozygous CCR5-Δ32/Δ32 CB donor unit are several logs easier, despite the cell dose limitations of CB products and the smaller sizes of CB inventories compared to bone marrow registries worldwide. In fact, the difficulties in the logistics and incredible cost barriers of screening tens of millions of adult donors for the CCR5- Δ32/Δ32 genotype cannot be over-emphasized. In contrast, with readily available archived samples and smaller inventories to screen, screening CB banks' products is an easier and less expensive proposition. Moreover, the fact that CB banks are cryopreserved physical inventories instead of a virtual database allows for easier construction of large banks of genotyped and infectious and genetic disease-screened samples, perpetually available for immediate shipping and transplantation with no possibility of donor refusal.

matched sibling donor, the patient received an allogeneic 4/6 HLA A/B/DR-matched unrelated donor CCR5-Δ32/Δ32 CB product from the StemCyte inventory, supplemented with purified CD34+ cells from a haploidentical sibling [95, 96]. By genotypic and phenotypic analysis, the Barcelona patient's HIV strain was CCR5-tropic, with 2.1 copies of replication competent HIV

200 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

Despite incredible worldwide attention and many attempts to replicate the success of the Berlin patient, why has there been no other successful cured long-term survivors reported to date? For one, HSCT is a highly risky procedure, with expected worse survival in advanced cases of malignancies. Malignancy is a feature which all of these seven cases share [91]. Four of the six cases expired because of or partially due to relapses from their malignant conditions. The other cases died from infection or pneumonia—all common causes of post-transplant mortalities. Only one case out of eight reported viral rebound, albeit with virus of a different tropism which already existed in the patient prior to transplantation, perhaps indicating that viral rebound

Currently, the most important barrier to having a large CCR5-Δ32/Δ32 adult donor or CB inventory is the expense and logistical difficulties of screening millions of adult donors or hundreds of thousands of CB archival samples. The 26,000 StemCyte CB units, 10,000 CB units from the M.D. Anderson bank and 8000 adult donors tested in the German Red Cross Donor Registry represent the largest repositories screened for CCR5-Δ32/Δ32 genotypes to date. As such, the current availability of HLA-matched CCR5-Δ32/Δ32 homozygous donors is still extremely limited. The frequency of homozygous CCR5-Δ32 is around 1% in Caucasians, even less in the Middle East and 0% or almost 0 in Africa and in Asian countries like Taiwan, China and Japan [75, 87]. The requirement for rigorous co-selection of both suitable HLA matches and CCR5-Δ32/Δ32 genotypes leads to difficulties in finding appropriate adult donors to treat HIV infection with hematopoietic stem cells from adult donors. Cord blood, however, can tolerate 1 or 2 HLA A/B/DR mismatches and does not necessarily need HLA matches for HLA C/DP/DQ loci; therefore, a CCR5-Δ32/Δ32 CB inventory gives rise to a higher probability of finding a suitable CCR5-Δ32/Δ32 CB unit for individual patients [87, 97]. Instead of needing to find a 10/12 HLA A/B/C/DR/DP/DQ match for homozygous CCR5-Δ32/Δ32 adult donors, the chances of a 4/12 A/B/C/DR/DP/DQ homozygous CCR5-Δ32/Δ32 CB donor unit are several logs easier, despite the cell dose limitations of CB products and the smaller sizes of CB inventories compared to bone marrow registries worldwide. In fact, the difficulties in the logistics and incredible cost barriers of screening tens of millions of adult donors for the CCR5- Δ32/Δ32 genotype cannot be over-emphasized. In contrast, with readily available archived

through tropism switch is not as easy as one might hypothesize [92].

 CD4 T cells and 303 copies per ml enumerated by single copy assay. After HSCT with CCR5-Δ32/Δ32 CB and haploidentical sibling donor purified CD34+ cells, plasma HIV DNA load was confirmed to be undetectable using ultrasensitive analysis. Upon reaching full chimerism with the CB donor, the patient's engrafted CCR5-Δ32/Δ32 CD4 T cells responded to proliferation and activation stimuli in vitro but was resistant to infection by the patient's viral isolate and laboratory strains. Unfortunately, death related to lymphoma progression prevented long-term monitoring of the patient's status; however, this case shows the potential promise and utility of CCR5-Δ32/Δ32 CBT in curing HIV if the patient was infected with CCR5-

per 107

tropic virus [96].

To explore the potential of CB stem cells as a more accessible source for curing HIV through HSCT, starting in 2001, working with City of Hope, Chow and Petz started screening for CCR5- Δ32/Δ32 genotypes of ∼18,000 Caucasian and ∼8000 Asian CB units and identified 134 Caucasian and 0 Asian CB units with the CCR5-Δ32/Δ32 genotype from StemCyte International Cord Blood Center [87]. During the last few years, additional CB inventories from other cooperating CB banks are being tested, and the CCR5-Δ32/Δ32 unit number has grown to ∼180 by 2013 [89] with the hope of increasing the inventory to at least 300 CCR5-Δ32/Δ32 CB units [87]. Studies have shown that the engraftment and survival of CBT recipient are highly dependent on the number of nucleated and CD34+ cells. The most commonly accepted threshold for TNC dose is 2.5 × 107 nucleated cells/kg recipient body weight, but this number may not be achieved by all CB units collected, especially for adult patients. Delayed engraftment and immune reconstitution are observed with CBT due to the low progenitor cell numbers in CB products. With a theoretical inventory of over 300 CCR5-Δ32/Δ32 cryopreserved units, working with the NMDP bioinformatics team, it has been predicted that the ≥4/6 HLA match rate would be 73.6% for Caucasian pediatric patients (younger than age of 16) at the minimal TNC dose of 2.5 × 107 nucleated cells/kg recipient body weight. Far lower rates at ≥ 4/6 HLA match are found for adult patients (16 years or older) of all race groups at this minimal TNC dose of 2.5×107 nucleated cells/kg recipient body weight – 27.9% for Caucasian, 2.7% for Chinese-American, 9.9% for African-American, and 14% for Mexican American. To overcome the problems of insufficient cell dose with CBT, double and sequential CBT methods have been developed to overcome this restraint, expand the access of CBT to patients and improve transplantation outcomes [98–100]. With the option of double CBT, the minimum number of nucleated cells necessary for successful transplant drops to a minimal TNC of 1.0  × 107 nucleated cells/kg for each CB unit and 2.5 × 107 nucleated cells/kg for the combined cell dose, thus allowing more patients, especially adult and heavier pediatric patients, to have the access to suitable CB donors [100]. According to Petz et al., the rate of finding 4/6 HLA-matched units can reach 85.6% for Caucasian pediatric patients and 82.1% for Caucasian adults, when only 1.0 × 107 nucleated cells/kg is applied as the selection criteria [87].

Unfortunately, the possibility of finding two 4/6 HLA-matched CB products for double CBT for a single HIV-infected patient out of a small 300 CCR5-Δ32/Δ32 CB unit inventory is also remote. Since that probability approximates zero, to reach a combined TNC of 2.5 × 107 nucleated cells/kg is essentially the same as just using a single CB unit with TNC of 2.5 × 107 nucleated cells/kg. As you can see from **Table 5**, as such, the chances of a double CBT with both CB units being homozygous for the CCR5-Δ32 mutation and with combined TNC ≥2.5  × 107 are expected to be similar to the probability of finding a single homozygous CCR5-Δ32/ Δ32 CB unit with TNC ≥2.5 × 107 projected by Petz et al. [87].


**Table 5.** Projected match rate with an inventory of 300 CCR5-Δ32/Δ32 cord blood units using double CCR5-Δ32/Δ32 cord blood transplantation strategy versus the CCR5-Δ32/Δ32 Cord blood + bridging CCR5 or CCR5/CCR5-Δ32 cord blood or haploidentical donor strategy.

Instead of homozygous CCR5-Δ32/Δ32 double CBT, we and others have proposed alternative "bridging" strategies. If a single CCR5-Δ32/Δ32 CB unit does not meet the ideal 2.5 × 107 nucleated cells/kg recipient body weight cell dose threshold, a second CB unit with either wildtype (WT) CCR5 or heterozygous CCR5/CCR5-Δ32 donor may be used in a double CB transplant ("the CCR5-Δ32/Δ32 CB + CCR5 CB OR CCR5-Δ32/Δ32 CB + CCR5/CCR5-Δ32 CB double CBT strategy"). Alternatively, it is possible to combine the first homozygous CCR5- Δ32/Δ32 CB product with a bridging haploidentical adult donor with wild-type CCR5 or heterozygote CCR5/CCR5-Δ32 genotype as a second graft to "bridge" the patient until the homozygous CCR5-Δ32/Δ32 CB unit has engrafted ("the CCR5-Δ32/Δ32 CB + haploidentical CCR5 donor OR CCR5-Δ32/Δ32 CB + CCR5/CCR5-Δ32 haploidentical adult donor strategy") [76, 91, 94, 101]. This was the strategy tried for the Barcelona and Dutch patients [93, 94, 96]. The hope is that the survival advantage of CCR5-Δ32/Δ32 CB against HIV infection and lysis of wild-type CCR5 or heterozygote cells would enable the homozygous CCR5-Δ32/Δ32 donor CB to prevail over the wild-type CCR5 or heterozygous CCR5/CCR5-Δ32 CB or haploidentical adult graft eventually. Since wild-type CCR5 or heterozygote CCR5 donors are readily available, this strategy will yield far higher probability of finding a match since only TNC ≥1.0  × 107 /kg is required for the first homozygous CCR5-Δ32/Δ32 CB product. As seen in **Table 5**, the effect of just requiring TNC ≥1.0 × 107 /kg for the first homozygous CCR5-Δ32/Δ32 cord blood product resulted in a much higher match rate for adult patients—27.9 to 82.1% for Caucasians, 2.7 to 13.9% for Chinese Americans, 9.9 to 31.6% for African Americans and 14 to 48.9% for Mexican Americans. Moreover, if this strategy is combined with Chow's MaxCell CB processing technologies and no-wash direct infusion strategy, then the probability of finding a CCR5-Δ32/Δ32 CB product with higher cell dose further increases [1, 8].

**Double CCR5‐Δ32/Δ32 CBT CCR5‐Δ32/Δ32 CBT + WT CCR5 OR CCR5/**

**Pediatric patients @ combined CCR5‐ Δ32/Δ32 CB TNC ≥2.5 × 107**

6/6 HLA A/B/DR matches ∼0.01% ∼0.01% 0.09% 1.01% ≥5/6 HLA A/B/DR matches ∼4.5% ∼10.6% 10.7% 10.8% ≥4/6 HLA A/B/DR matches ∼27.9% ∼73.6% 82.1% 85.6%

≥4/6 HLA A/B/DR matches ∼2.7% ∼12.3% 13.9% 15.7%

≥4/6 HLA A/B/DR matches ∼9.9% ∼28.6% 31.6% 34.1%

≥4/6 HLA A/B/DR matches ∼14% ∼44.1% 48.9% 52.5%

**Table 5.** Projected match rate with an inventory of 300 CCR5-Δ32/Δ32 cord blood units using double CCR5-Δ32/Δ32 cord blood transplantation strategy versus the CCR5-Δ32/Δ32 Cord blood + bridging CCR5 or CCR5/CCR5-Δ32 cord

Instead of homozygous CCR5-Δ32/Δ32 double CBT, we and others have proposed alternative "bridging" strategies. If a single CCR5-Δ32/Δ32 CB unit does not meet the ideal 2.5 × 107 nucleated cells/kg recipient body weight cell dose threshold, a second CB unit with either wildtype (WT) CCR5 or heterozygous CCR5/CCR5-Δ32 donor may be used in a double CB transplant ("the CCR5-Δ32/Δ32 CB + CCR5 CB OR CCR5-Δ32/Δ32 CB + CCR5/CCR5-Δ32 CB double CBT strategy"). Alternatively, it is possible to combine the first homozygous CCR5- Δ32/Δ32 CB product with a bridging haploidentical adult donor with wild-type CCR5 or heterozygote CCR5/CCR5-Δ32 genotype as a second graft to "bridge" the patient until the homozygous CCR5-Δ32/Δ32 CB unit has engrafted ("the CCR5-Δ32/Δ32 CB + haploidentical CCR5 donor OR CCR5-Δ32/Δ32 CB + CCR5/CCR5-Δ32 haploidentical adult donor strategy") [76, 91, 94, 101]. This was the strategy tried for the Barcelona and Dutch patients [93, 94, 96]. The hope is that the survival advantage of CCR5-Δ32/Δ32 CB against HIV infection and lysis of wild-type CCR5 or heterozygote cells would enable the homozygous CCR5-Δ32/Δ32 donor CB to prevail over the wild-type CCR5 or heterozygous CCR5/CCR5-Δ32 CB or haploidentical adult graft eventually. Since wild-type CCR5 or heterozygote CCR5 donors are readily available, this strategy will yield far higher probability of finding a match since only TNC ≥1.0 

/kg is required for the first homozygous CCR5-Δ32/Δ32 CB product. As seen in **Table 5**,

blood product resulted in a much higher match rate for adult patients—27.9 to 82.1% for

/kg for the first homozygous CCR5-Δ32/Δ32 cord

**Adult patients @ combined CCR5‐ Δ32/Δ32 CB TNC ≥2.5 × 107**

202 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

Caucasians

Chinese American

African American

Mexican American

× 107

Adapted from Petz et al. [87].

blood or haploidentical donor strategy.

the effect of just requiring TNC ≥1.0 × 107

**Donor Transplant**

**Adult patients @ CCR5‐Δ32/Δ32 CB TNC ≥1.0 × 107**

**CCR5‐Δ32 CBT OR Haploidentical Adult**

**Pediatric patients @ CCR5‐Δ32/Δ32 CB TNC ≥1.0 × 107**

Instead of having HIV-infected patients search for matched donor CCR5-Δ32/Δ32 CB, the more efficient strategy may be for the limited CCR5-Δ32/Δ32 donor inventory to find matched HIV patients who have concomitant transplant indications [1]. In this scenario, instead of having HIV-infected patients searching for CCR5-Δ32/Δ32 donors, it is possible to reverse the process and have the HLA type CCR5-Δ32/Δ32 CB or adult donors database, look for HIV-infected individuals with transplant indications who have suitable HLA matches among the CCR5- Δ32/Δ32 inventory. The math of a patient looking for a graft and a graft looking for a patient is the same, just in the opposite directions; however, this allows targeted CCR5 genotype screening of HIV patients who are in need of a HSCT [1].

Overall, HSCT has the potential to accomplish functional or sterilization cure of HIV patients and would be especially valuable to HIV patients with hematological malignancy, which may cure both indications with the same HSCT. Cord blood could be a more accessible alternative to HLA-matched adult donor for curing HIV infection through HSCT, even though more clinical trials on HIV patients would be necessary to establish the efficacy in eradicating HIV infection for both adult donor HSCT and CBT. Due to the difficulties of co-selecting for units with ≥4/6 HLA A/B/DR matches, suitable nucleated or CD34+ cell doses and the CCR5-Δ32/ Δ32 genotype, innovative strategies such as (1) double grafts combining a homozygous CCR5- Δ32/Δ32 CB with either another wild-type or CCR5/CCR5-Δ32 heterozygote graft, (2) combined with MaxCell CB processing and thawing technologies that yield higher cell doses and (3) the employment of the reverse strategy of looking for matched HIV patients in need of HSCT, can be helpful in expanding the utility of the CCR5/CCR5-Δ32 CB inventory for viable transplantation. Actively screening for more CCR5-Δ32 homozygous CB units, establishment of more comprehensive HIV-infected patient HLA type database and the application of double CBT or bridging strategies would eventually allow more HIV-infected patients find suitable units for transplantation.
